S. Arr. J. Bot. 1997, 63(6) 345-350 345

Somatic embryogenesis of two indigenous South African spp. (H. limifolia and H. koelmaniorum)

D.J. Mycock1*, M.P. WatF, K.F. Hannweg2 , K. Naickerl, M. Makwarela2 and P. Berjak2 1Department of Botany, University of the Witwatersrand, Private Bag 3, Wits, 2050 Republic of South Africa 2Centre for Indigenous Use Research, Department of Biology, University of Natal, Private Bag X10, Dalbridge, 4014 Republic of South Africa

Received 18 March; revised 12 June 1997

The endangered, indigenous succulents, Haworthia koe/maniorum and H. limifolia, have been micropropagated using somatic embryogenesis. The somatic embryo induction medium contained MS macro- and micro-nutrients (MS}, 20 g 1·1 sucrose and 10 g 1·1 agar, pH 5.6. For H. limifolia the following plant growth regulators were tested: 2,4-D (1, 2 and 5 mg 1·\ picloram (1 and 2 mg 1·1} and 0.25 mg 1·1 kinetin plus 1 or 2 mg 1·1 picloram. Direct somatic embryogenesis occurred best from explants (apical one third of leaf) exposed to 2 mg 1'1 2,4 D. However, when the embryoids were transferred to plant growth-regulator-free medium they did not germinate but rather de-differentiated to callus and then underwent secondary embryogenesis. Hyperhydricity of the secondary embryoids was obviated by culturing in the presence of silica gel, and material treated in this manner differentiated into plantlets, which could be hardened. For H. koe/maniorum 2 mg 1·1 picloram and 0.25 mg 1"1 kinetin were also added to the embryo induction medium and the effect 1 of ABA on embryoid induction was tested at 0.1, 0.2 and 0.3 mg 1· . Somatic embryoids, which could be germinated and hardened, developed directly from somatic cells of the leaf explant when the tissues were exposed to either 0.2 or 0.3 mg 1·1 ABA.

Keywords: Haworthia, micropropagation, somatic embryogenesis, hardening-off.

• To whom correspondence should be addressed.

Introduction this is a slow process which is exacerbated by the low number of The genus Haworthia (Liliaceae) is native to South Africa and offshoots produced by each plant (Huxley 1979; Sheat 1987; Namibia, and is characterised by tremendous morphological Pienaar 1992). Consequently, micropropagation can be consid­ variation in the field. Due to the fact that early taxonomists ered n viable and attractive alternative means of mass multiplica­ described individual , the of the genus has been tion oftliese endangered . equivocal; for instance, over 400 names have been used at the To date, there have been no published reports on the in vitro infra-generic levels (Court 1981 ). The genus is largely restricted propagation of H. koelmaniorum and H. lim((olia, but several to the southern Cape province, although H. limifolia occurs in papers describing micropropagation procedures for other Hawor­ Mozambique, Swazi land, Mpumalanga and KwaZulu-Natal, H thia species have appeared in the literature (Table 1). Although koelmaniomm in the Northern Province and H. arachnoidea in Bey! and Sharma ( 1983) obtained H. fasciata plantlets via Namibia. Haworthia species must therefore be seen in the con­ somatic embryogenesis, all of the other reported procedures text of geographical distribution and field relationships with describe direct or indirect organogenic regeneration. Somatic other species (Court 1981 ). embryogenesis is a potentially valuable tool in germplasm con­ Hmvorthia species tend to grow in pockets of shallow soil in servation as the material is genera lly most su itable for in vitro open to closed bushland (Boyd 1994). Intensive agricultural storage. For example, somatic embryos may be cryopreserved practices have resu lted in the fragmentation and isolation of pop· (Engelmann et at. 1995; Mycock et a/. 1995) or encapsulated ulations to sandstone ridges. This, combined with competition (Bapat & Rao 1988) and in this manner be stored until the need from invasive species, has had n strong negative effect on species arises for their retrieval and in vitro regeneration and mul tiplica­ population stability. Survival of the genus, and in particular the tion (McKersie 1995). two species H. koelmaniorum and H. limifolia, is also threatened The ultimate aim of this work is the multiplication for field by intensive utilisation in traditional medicine (Cunningham establishment and the in vitro storage of H. koelmaniorum and H. 1988). Hauwthia spp., referred to as 'uMathithibala', are sought limifolia genotypes. Consequently, our strategy towards the first after by local traditional healers because of their all eged power­ objective was to test and adapt, if necessary, the method of Bey I fu l healing properties in the treatment of constipation, diarrhoea, and Sharma ( 1983) for these species. The protocol recommended dysentery, haemo rrhoids, indigestion, nausea, stomach ache and by those authors also offers the advantage in that it uses leaf tis­ worms (Hutchings 1989). In addition, when the leaves are strung around the neck, they are reported to create an euphoric feeling sue as the initiating cu lture explant, th ereby being non-destruc­ and to ward off evil spirits (Hutchings 1989). As a result of their tive to the limited (endangered) material that is available endangered status the two species were declared as protected throughout the year. The investigations were duplicated in two under the Transvaal Nature Conservation Ordinance of 1983, laboratories to confirm the reproducibi li ty of the results. 86- 88 schedule 11. Both H. koelmaniorum and H. limifolia produce copious Materials and Methods amounts of seed, but these must be propagated whilst fresh as Plant material viabi lity declines within three months (unpublished data; see also Soi l-grown adult H. limifolia and H. koelmaniorum plants were Huxley 1979). The two species may also be vegetatively propa­ obtained from Si!verglen Nursery (Durban) and the National Botani­ gated via offsets from the parent plant or from leaf cuttings, but cal Institute (Pretoria), respectively. The plants were maintained at 346 S. Afr. J. Bot. 1997, 63(6)

Table 1 Response of different explant types from various Haworthia species to in vitro culture Species Explant Mode of Regeneration References

Haworthia spp (I 0) Gynoecium Indirect organogenesis (shoots) Kaul & Sahbamal ( I 972; 1975)

Haworthia spp (5) Middle section ofleaf Direct organogenesis (plantlets) Rogers ( 1993) Direct and indirect organogenesis H. arachn01dia Perianth (shoots and flowers) Konishi ''/a/. ( 1982) Direct and indirect organogenesis H. qmbiforrnis Perianth (shoots and flowers) Konishi eta/. (I 982)

I f. fascia/a Basal section of leaf Indirect embryogenesis (plantlets) 13cyl & Sharma ( I 983)

H. p/anifolia Leaf sections Indirect organogenesis (plantlets) Wessels ct a/. ( 1976) Direct and indirect organogenesis H. turg1da pal/idifolia Ovary (plantlets) M

14/ 10 h light/dark photoperi od. 600 ~t E m2 sec· 1 and 18-25°C night/ containing potting soil : (ii) as (i} hut sealed with pierced transparent day temperatures. plastic; (iii) in pl astic-covered seedling trays containing moistened potting soil and mist-sprayed dai ly; (iv) in plastic-covered seedling Culture conditions trays containing dry potting soil and mist-sprayed daily: (v) in The apical two-thirds of mature leaves were excised from the parent uncovered seedling trays containing dry potting soi l and plant. The material was surface sterilised for 20 minutes in I% (v/v) mist-sprayed daily: (vi) in small pots (75 x 75 x 75 mm) containing (H. /imifo/ia) and 2% (v/v) (H. koelmaniorum) sodium hypochlorite moist potting soil, sealed in plastic bags and mist sprayed daily. solution containing I drop Tween 20 per I 00 ml of sterilant, fol­ Hardening of H. koe/maniorum plantlcts was achieved using small lowed by three rinses in sterile water. For H. limifolia only the top pots sealed in plastic bags (rcscalable) which were opened and mist half of the excised leaf. and, for H. koelmaniorum, the entire portion sprayed weekly. of the ~ t e rili sed leaf material was used for culture. The material was Microscopy, photography and data analyses then cut into 5 mm 2 segm ~: nt s. The somatic embryo induction medium contained Murashige and Embryoids were examined at the light microscopt: lt:vel and photo­ Skoog ( 1962) macro- and micro-nutrients (MS), vitnmins and amino graphed using a WILD M3 stcreomicroscupc with photoautomat acids according to the formulation of Bey I and Sharma ( 1983). 20 g MPS 55 system (Durban) or WILD 420 stcreomicroscope (Wits). J·1 sucrose and 10 g J· 1 agar, pH 5.6. The following combinations of Large subjects were photographed using a Nikon FM2 camera with a growth regulators '.'iere tested: Mikro Nikkor 60 mm macro lens. Data were assessed using one-way (i) for H. limifolia, I , 2 and 5 mg 1·1 2,4-dichlorophenoxy-acetic acid ANOVA [p < 0.05] (Statgraphics Plus, 1993 Edition). Alphabetical (2,4-D), I and 2 mg J·l 4-amino 3,5,6-trichloropicolinic acid (piclo­ values were assigned to the means. with different letters indicating significant differences. ram), 0.25 mg J·1 6-furfurylaminopurine (kinetin)+ 1 mg J·1 picloram 1 and 0.25 mg J· kinetin + 2 mg J·l picloram. Results and Discussion (ii) for H. koelmaniorum. 0.25 mg J·1 ki_netin + 2 mg J-1 picloram + H. limifolia- Media composition 0.1 mg J·1 abscisic acid (ABA), 0.25 mg J·1 kinetin + 2 mg 1·1 piclo­ ram + 0.2 mg J·1 ABA and 0.25 mg 1·1 kinetin + 2 mg 1·1 picloram + Bey! and Sharma (1983) reported that picloram outperformed 2,4-D as the auxin source for both the induction and final yield of 0.3 mg J·1 ABA. After 14 weeks in the dark at 25°C, the explants were transferred somatic embryoids of H. .fascia/a. However, in the present study, to regeneration medium (somatic embryo induction medium without significantly more H. lim[folia explants exhibited an embryo­ growth regulators), a 16/8 h light/dark photoperiod, 200 !-!E m2 sec-1 geni c response to 2,4-D (at any of the concentrations tested) than and 25-27°C for approximately six weeks. to picloram (Table 2). Although the addition of0.25 mg J·1 kine­ tin to the picloram-enriched media, as recommended by those Studies on Hyperhydricity authors, increased the production of somatic embryoids, the Hyperhydricity was only observed with H. /imifo/ia plantlets. After response remained significantly lower than that for any 2,4-D 4 weeks on the regeneration medium plantlets were transferred to treatment. The best embryogenic response for H limifolia was fresh regenerati on media containing one of the following: 4 or 8 g obtained from 2,4-D at a concentration of2 mg 1·1• 1 J·l Gelrite (replacing agar), 12 g J-l agar, 0.25 or 0.5 mg J· CoCI2, 1 1 1 2 H. limifolia - Development and hardening 1.25 g J· NH/. 1.6 mg J· K + (as K 2S04), 0.88 or 1.22 g J· Ca + (as 1 Within 7- 14 days of transfer to the optimum embryoid induction CaS04), 0.06 or 0.12 g J· phloroglucinol (DeBergh et a/. I 992). In another treatment 6 g of silica gel in a small alum inium foil con­ medium, a clear-crystalline callus developed over the H. /imifo­ tainer was placed inside each culture vessel (65 mm diameter x 70 lia cut surfaces. These observations we re in accordance with mm height). which contai ned 30 ml medium. The silica gel was those of Bey! and Sharma ( 1983) who reported call us formation replaced every second day. Each experiment was conducted three in H fascia/a within two weeks. After six weeks in culture yel­ times using 30 plantlets. low embryoids could be distinguished between the two epider­ mal surfaces of the leaf explants (Figure I). It is important to Hardening-off note that the surface callus was not associated with these struc­ H. limifolia plantlets (30 plantlets and three replicates) were hard­ tures, thus suggesting that embryogenesis in H. limifolia pro­ ened off and maintained for 8 weeks under the different conditions ceeded directly from the somatic cells (e.g. mesophyll cells) of listed below: the cultured explant. Subculturing the explants onto medium (i) In closed sterile culture bottles (65 mm diameter x 70 mm height) devoid of 2,4-D did not result in the formation of plant lets, but S. Afr. J. Bot. 1997. 63 (6) 347

Table 2 Quantitative and qualitative estimation of Haworthia explants exhibiting embryoid formation after 12-14 weeks in culture. (Values with the same letter are not significantly different at P = 0.05), + weak response, +++ strong response, NT not tested Explant Growth Regulators 1

Leaf section 2.4-D Pic1oram Kineti n! 21 & Picloram Kinetin!21, PicloramPI & AI3A

Base Mid Tip 2 5 2 2 0.1 0.2 0.3 II. Fascia/a (Bcyl & +++ + + + + NT NT +++ NT +++ NT NT NT Sharma I 9&3)

II. linujiJ!i a + ++ +++ 6J.9C 7 3.3~ 63. 3r 15.4" 28.J C 23.5b 32.3d NT NT NT

II. koelmmziorum + ++ +++ NT NT NT NT NT NT 52.0b 39.0' 56.0b 58.0h

(I) mg t· 1 (2) 0.25 mg t·1 kineti n (3) 2mg 1' 1 Piclormn rather induced callus proliferation from the embryoids followed (Hedegus & Phan 1983) not only maintained the hyperhydric by secondary embryogenesis [1 2- 14 weeks after culture initia­ state but appeared to be assoc iated with increased plant mortal­ tion] (Figure 2). The embryoids produced in this indirect manner ity. The water potential of a medium al so has an effect on hyper­ germinated and developed into plantlets (Figure 3). hydricity, thus the addition of extra or use of a different One of the main problems encountered in the micropropaga­ so lidifying agent can be advantageous (George 1996). Replacing ti on of H. limifolia was the loss of plantlets (up to 60%) because agar with Gelrite (6 or 8 g ]·1) did not improve the hyperhydric of hyperhydricity. Healthy plantlets were identified as being state, but elevation of the agar concentration to !2 g ]·1 appeared opaque and dark green (Figure 4), whereas hyperhydric plantlets to reduce the condition, as plants subcultured onto thi s more were 'glassy ' and in extreme cases translucent. Hyperhydricity solidified medium appeared less translucent and green er. This results fro m metabolic perturbations induced by the combined di fference between the two solidifyi ng agents may be due to the action of both the physical and chemi cal nature of the culture fact that water in a Gelrite gel is only lightly bound, compared environment (DeBergh et a/. 1992; Geo rge 1996). Avoidance or with water in an agar gel (Scherer 1988). Nevertheless, when reversal of the condition can therefore be achi eved by manipul a­ plantlets were cultured in the presence of sili ca gel, whi ch pre­ tion of the culture medium compos ition, changes in the environ­ sumably lowered the re lative humidity in the culture vessel (RH mental conditions and thus quality of the cultured pl ants values not tested), they had a norm al appearance and could be (DeBergh eta/. 1992). Changes in the chemical composition of easily hardened off (see below). The most appropriate hard en­ the medium, i.e. increases in K', CoCI2 and Ca2 • concentra ti ons ing-off procedure was the transfe r of the pl antlets into seedling and a decrease in NH4 • concentrati on, as recommended by trays containing moistened soil (Table 3 and Figure 5). A twi ce Beauchesne (198 1) , Letouze and Daguin (1983) and Ziv et a/. daily mist-spray was required. to maintain humidity and prevent ( 1987) did not have any effect on reducing or reversing the hype­ rhydric state. Furthermore, the addition of phloroglucinal

Figure 2 Haworthia limifolia. The primary embryoids de-differ­ Figure 1 Haworthia limifo/ia. Yellow somatic embryo ids devel­ entiated into callus and then differentiated into secondary embryoids oped di rectly from the non-epidermal tissues of the leaves. when cultured on nutrient medium devoid of 2. 4-D. 348 S. Afr. J. Bot. 1997, 63(6)

formation in H. kvelnumiorum (Tabl e 2) but at concentrations of 0.2 and 0.3 the formation of embryoids was above 50%. More importantly the somatic embryoids for med in the presence of these higher leve ls of ABA developed into plantlets within 4-6 weeks of transfer to regeneration medium. whereas the non ABA treated embryo ids did not develop fu rther (see above).

H. koe/maniorum - Development and hardening In H. koe/maniorwn cultures, a white friable, surface callus \Vas observed wi thin 7 days of cul ture initiation and afte r three weeks the callus covered the enti re explant. Since thi s phenomenon was observed in all media variations, it was presumed to be du e to the presence of both the auxin (picloram) and cytokinin (kinetin) which were common to all the media. After eight \veeks in cul­ ture (on the media containing th <.: high~:r concentrati ons of ABA) the callus had browned and presu mab ly died. but ye llow somati c embryoids which had developed directly from the somatic cells of the explant were observed in the brown callus mass. Subcul ­ turing the explants onto the same medium for a further 6 weeks all owed for continued generation of additional embryo ids but not their germination. However. when the explants were subse­ quently transferred to a med ium devoid of plant growth regula­ tors, the embryoids germinated into plantlets. Thus somatic Figure 3 Hmvorthia /imifolia. The secondary cmbryoids devel­ embryogenesis of this species was different from that of II limi­ oped into plantlcts with distinct shoot and root axes. .fo/ia in that it did not progress through two cycles, i.e. secondary embryogenesis, but rather required the maturation influence of the plantlets from desiccating. Plants in dry soil had a slightly ABA (George 1996) in the direc t development of primary lower survival rate (70.6%). In contrast, plantlets grown in soil in embryoids. The efficacy of ABA in the conversion (maturation) sealed or partially sealed culture bottles exhibited much lower of somati c embryos has IH.:en conlirmed in a number of other levels of survi val. this was presumably the resu lt of plantlet species e.g. Daucus carota and I'JC

Table 3 SuNival of in vitro generated H. limifolia plant­ lets under various hardening treatments Percentage Survival (4 Treatment weeks)

i) Sterile glass bottles (rlastic lids) 9.7' ii) Sterile glass bottles (aerated lids) 10.2h iii) Seedling trays (moist condi- tions-covered) 50.3° iv) Seedling trays (dry conditions-cov- ered) 62.3d v) Seedling trays (dry conditions + mist spray-no cover) Figure 4 Haworthia limifolia. Non-hyperhydric, healthy plantlcts vi) Small pols (moist conditions + mist were characterised by dark green lcnves. spray-covered) 70.6< S. Afr. J. Bot. 1997. 63(o) 349

stand desiccation (George 1996). Consequently the inclusion of this plant growth regulator in the H. koelmaniurum micropropagtion procedure, here used to mature the embryoids, may also prove useful in the preparation of the tissues for cryostorage.

Acknowledgements The authors wish to thank the FRD, the University of Natal Research Fund and the University of the Witwatersrand Research Council for funding this collaborative research project.

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