Mist House Research Articles

Performance of Guava Stem Cutting – A Review of Successful Though Non-commercial Propagation Method

Propagation of Guava (Psidium guajava L.) can be successfully performed by stem cutting as demonstrated by many researchers time to time. Though, guava is found hard to root, use of root inducing hormone (IBA, IAA, NAA etc.), rejuvenating techniques (fungicide, wounding, Girdling etc.), growth supporting media (silt, Sand, cocopeat, vermicompost, vermiculite etc.) and suitable protected environment (mist house, polyhouse, net house etc.) ease the rooting of cutting. Despite of the fact that propagation through cutting is easiest and most convenient method of vegetative propagation to regenerate numerous planting materials in short duration, wedge grafting is still recommended as commercial method of propagation in Guava. This scenario raises the question on potentiality of stem cutting as viable propagation method. This might be due to complex mechanism behind guava stem cutting propagation which results in inconsistent and highly variable results. Moreover, propagation involves multiple factors which have combined influence on the performance of stem cutting viz., preparation of cutting, preconditioning treatment, season of taking cutting, type of cutting, chemical treatment, growing environment, growing media and container. Therefore, to establish guava stem cutting as viable and commercial propagation technique, in depth study of these factors is vital. This paper briefly discusses the research so far has done with relevance to various factors influencing performance of guava stem cutting while indicating possible future research directions for more encouraging and consistent results.

Seed Storage and Germination Performance in Knema attenuata - an Endemic Medicinal Tree of Western Ghats, India

Detailed study on seed storage and germination trailed in Knema attenuata (Wall. ex Hook. f. & Thomson) Warb.– the IUCN Red Listed ‘least concern’ medicinal tree species revealed that seeds were of recalcitrant nature. Viability of the seeds could be maintained for a longer period of up to 6 months with 47% Moisture content (mc) when kept in closed polycarbonate bottles at seed bank condition [20±20C Temp. and 40% Relative Humidity (RH)]. The 55% seed germination under normal climatic condition could be enhanced to a much higher percentage (75±5) inside the mist house chamber (34±30C Temp. and 70-80% RH).

Early growth and graft success in macadamia seedling and cutting rootstocks

Rootstocks play a vital role in growth and productivity of tree crops through water and nutrient translocation and signal transduction to the scion. Early vigour and graft compatibility of rootstocks are important for further growth and development of the scion, and hence for productivity. This study aimed to identify the variability of early growth and graft success of cuttings and seedlings of different species, cultivars and breeding lines of macadamia that will be subsequently screened in a macadamia rootstock field trial. The experiments were conducted in a mist house at Maroochy Research Facility, Nambour. The experiments comprised macadamia seedlings and cuttings of eight commercial cultivars, fifteen breeding lines, and different sources of wild germplasm of Macadamia tetraphylla, M. jansenii and M. ternifolia. All the existing cultivars and breeding lines in the study are derived from M. tetraphylla and/or M. integrifolia species. There was significant variation among the genotypes in cutting survival and growth rates for both seedlings and cuttings. The study identified a breeding line (BHI2) which produces strong cutting root systems with better striking rates (100%) than existing rootstock cultivars. Cutting survival rate was greatest in standard macadamia rootstocks (79%) and least in M. ternifolia (46%). Growth rate varied depending on the genotype and the method of propagation. ‘H2’ seedlings were greatest for height increase rate, while cuttings of M.jan3, M. ter1, M.ter3, BHI1, BHI2, BHY1, ‘A268’, ‘A4’, ‘D4’ and ‘Daddow’ showed greater growth in height than ‘H2’. Both seedlings and cuttings of BHI2, cuttings of BDW4, M.ter1, M.jan3, BHY1, and seedlings of M.tet2 had greater stem diameter increase rate than ‘H2’. Among groups, standard rootstocks had the greatest rate of increase in height and breeding progeny of high index value had the greatest rate of stem diameter increase. Graft success was significantly higher in seedlings than cuttings. Cuttings of BHI2, ‘D4’ and ‘Daddow’, and seedlings of BDW5, M.tet2, BHI3 and BHY1 outperformed ‘H2’ for graft success. Results from this study suggest that initial rooting in cuttings can be an indicator of graft success. After planting the grafted tree, the relationship of the pre-grafting characteristics of the rootstock with the performance of the scion can be used as tools of identifying high performing rootstocks in the macadamia breeding program.

IN VITRO PROPAGATION OF SHOOTS AND CALLUS INDUCTION OF GYMNEMA SYLVESTRE R. BR. “AN IMPORTANT ANTI-DIABETIC PLANT”

Objective: The objective of this research was to establish and develop a protocol for the mass multiplication and callus induction of an anti-Diabetic plant-G. sylvestre R. Br.Methods: Sterilized explants (Nodal segment and leaf) were used for the initiation of culture. They were cultured on MS medium supplemented with a variety of PGRs (BAP, Kn, IBA, 2,4-D) individually or in combinations.Results: The induction of multiple shoots from nodal segments were highest in MS medium supplemented with 2.0 mg/l Kn and in BAP Maximum shoots were obtained on MS medium fortified with 1 mg/l BAP. For rooting different concentration of IBA were used and highest rooting was recorded on MS medium supplemented with 2.0 mg/l IBA. The rooted Plantlets were hardened initially in culture room conditions and then transferred to mist house. Leaf petiole explants were used for the purpose of callus induction. Best growth was observed in MS medium supplemented with 2,4-D. 1.0 mg/l 2,4-D+0.5 mg/l BAP, 1.0 mg/l 2,4-D+1.5 mg/l Kn.Conclusion: The results obtained in this research work clearly indicated that Kn is a better choice than BAP for the culture initiation. 2 mg/l IBA was proved best for root induction. For callus induction, 1 mg/l 2,4-D gave good results and when callus was sub-cultured on 2,4-D with BAP or Kn then 1.0 mg/l 2, 4-D+1.5 mg/l Kn proved best for mass propagation of callus.

Embryogenesis followed by enhanced micro-multiplication and eco-restoration of Calamus thwaitesii Becc.: an economic non-wood forest produce for strengthening agroforestry system

The present study is focussed on development of a high-frequency micro-multiplication system in Calamus thwaitesii, through somatic embryogenesis from immature zygotic embryos cultured in Murashige and Skoog (MS) medium supplemented with 31.68 µM 2,4-dichlorophenoxyacetic acid (2,4-D). The semi-friable calli when cultured in the same medium augmented with 2.22 µM 6-benzylaminopurine (BAP) and 1.07 µM α-naphthalene acetic acid (NAA) induced ~ 12 discrete globular embryoids in 6 weeks. The isolated embryoids in hormone-free media yielded 65% plantlets. Furthermore, embryoids and axenic shoots exhibited maximum shoot induction in medium supplemented with 0.45 µM Thidiazuron (TDZ). The shoot initials after subculture in media supplemented with 1.78 µM BAP and 0.45 µM TDZ produced shoot proliferation followed by elongation in basal medium. The elongated shoots produced roots in media supplemented with 16.11 µM NAA. With this established protocol, ~ 5940 rooted plantlets could be harvested after 40 weeks from a single embryoid. Genetic stability analysis of the plantlets using inter simple sequence repeat markers recorded only 0.05% genetic polymorphism. The plantlets were hardened in a mist house for 8 weeks, and then to 50% shade house for another 16 weeks, and the well-established 6-month-old nursery plants, reintroduced to selected forest segments, exhibited 86% field establishment even after 3 years of observation. Thus, the mass multiplication system developed could be a breakthrough for large-scale multiplication of C. thwaitesii to ensure continuous supply of quality planting material to the cottage industry through the development of agroforestry systems. Furthermore, the in vitro culture system developed here can be replicated for research activities related to the long-term–short-term conservation, micro-multiplication and sustainable utilization of rare, endangered, endemic, monopodial/single stemmed rattan palms.

Somatic Embryogenesis on Plumule and Radicle Explants Obtained from Warm Water Hydroprimed Wheat (Triticum aestivum L.) cv. Kunduru and cv. Cakmak Seeds

Advances in wheat biotechnology and in vitro somatic embryogenesis (SE) provide new openings to faster application of these techniques in functional genomic studies, genetic engineering and plant breeding. This study reports in vitro SE using plumule and radicle explants of two wheat cultivars Cakmak and Kunduru on MS induction medium amended with varying concentrations of 2,4-D. Both plumule and radicle explants were regenerative and induced variable number of somatic embryos per explant. Explants and treatment methods affected in vitro SE irrespective of the genotypes used in the study. Although 100% SE was noted on both explants of two cultivars, plumule explants of both genotypes induced heavy and larger friable calli followed by somatic embryogenic calli in terms of weight. Plumule explants also induced large numbers of shoot buds and somatic embryo-induced maturing shoots per explant. The in vitro raised plantlets were successfully rooted followed by hardening and acclimatisation in pots containing peat moss in mist house. After 7 days from weaning, these plants were transferred to a shade house to grow and set flowers. Water spray containing 0.50 μg of NPK (1:1:1) was given at intervals of 4 d to enhance success rate of acclimatised plants. Success rates of 96 and 80% were noted on plumule and radicle-induced plants, respectively. It is further concluded that use of this novel methodology would favorably facilitate its use in genetic transformation and functional genomic studies.

Standardization of in Vitro Regeneration Protocol for Conservation of Shirui Lily (Lilium Mackliniae)-An Endangered Heritage Flower under Changing Climatic Conditions

Shirui lily (Lilium mackliniae), an endangered Asiatic lily species, hails from the Shirui peak of Siroi hill range in Ukhrul district of Manipur, India. This rare species is now at the verge of its extinction due to uncontrolled trampling and plucking by the human intrusions and extensive slash and burning cultivation in this region, as a result of which, it has been recorded in the ‘Red Data Book’ of ‘Indian Plants: Volume I’. Conservation of this endangered species under changing climatic conditions is a great concern for the researchers. An attempt has been made in the present investigation to establish an efficient in vitro regeneration protocol in L. mackliniae for its conservation. Callus cultures were established in MS medium containing different concentrations of 2,4-D (1-2mg/l). The regeneration ability in friable callus was 12 fold higher as compared to the compact callus. Direct regeneration and somatic embryogenesis was achieved in friable callus at 6-8 weeks of subculture in MS with benzyladenine (BA, 1.0mg/l). Multiple shoot proliferation and rooting were achieved in MS+0.5mg/l BA and 0.5mg/l napthalene acetic acid (NAA), respectively. Hardening under mist house resulted in higher survivability (85%) over poly house (62.2%) and shed net (55.5%). The in vitro plantlets were evaluated under different temperature and moisture regimes. The first time report of in vitro regeneration protocol will be helpful to augment breeding strategies in this rare endangered species for wide adaptation under changing climate conditions.

Micro-cloning of an economic rattan palm Calamus thwaitesii for eco-restoration programme

Axillary shoots were induced from shoot tip of Calamus thwaitesii suckers on Murashige and Skoog medium supplemented with 0.4 mg/L N6-benzylaminopurine and 0.1 mg/L each of thidiazuron and α-naphthaleneacetic acid (NAA). The shoots initiated were subcultured to fresh media of the same composition for shoot multiplication and multiplied shoots were transferred to half strength MS hormone-free media for shoot elongation. The elongated shoots (∼5cm) were then re-cultured to the media supplemented with 3.0 mg/L indole-3-butyric acid/4.0 mg/L NAA to raise plantlets which were subsequently analysed for genetic fidelity using inter simple sequence repeat markers. Out of 183 bands scored, 178 bands were monomorphic indicating 97.2% similarity. The observed low level of polymorphism between genotypes supports genetic consistency of these micro-clones that are likely to be genetically true to their parental origin. The clones thus obtained were hardened in the specially fabricated mist house at 29 ±2°C and 80±5% relative humidity for 3 months followed by shifting to green house for another 3 months of nursery establishment. The established plants when reintroduced to the selected forest segments of the Western Ghats, Kerala (India) showed 79.3% survival rate after 2 years of field transfer. The viable and highly reproducible in vitro cloning protocol demonstrated here for the first time can be used for the production of elite female clones for aforestation activities and sustained delivery of high quality raw materials to cane processing units for strengthening cane industry.

In vitro Regeneration of an Endangered Medicinal Plant Withania somnifera using Four Different Explants

An efficient protocol was developed for highly regenerative capacity from leaf explant of Withania somnifera (L.) Dunal ? an endangered medicinal plant. Calli were regenerated from four different explants like leaves, cotyledons, hypocotyls and epicotyls. MS supplemented with different concentrations of 2,4?D, BAP and NAA were used. The calli (94.33 ± 1.20%) were obtained from the leaf explant in 2,4?D 3.0 mg/l. The highest number of multiple shoots (85.67 ± 0.88%) were obtained from the leaf callus at 4.0 mg/l BAP. Shootlets forming calli were transferred to the rooting medium containing 10.0 mg/l NAA to produce multiple roots (89.33 ± 0.88%). The regenerated rooted shootlets were transferred to small polythene bags, which contain a sterilized cow?dung, sand and red soil (1 : 2 : 3) and kept in a mist house. After acclimation in the mist house the regenerated plantlets were hardened in the greenhouse and transferred to soil, which showed 85% survival rate. This new protocol was standardized for easy mass propagation of W. somnifera using leaf explant.Plant Tissue Cult. & Biotech. 23(1): 79?85, 2013 (June)DOI: http://dx.doi.org/10.3329/ptcb.v23i1.15564

Conservation through in vitro propagation and restoration of Mahonia leschenaultii, an endemic tree of the Western Ghats

Conservation of Mahonia leschenaultii was achieved through multiple axillary shoot formation in single node cultures, in vitro and ex vitro rooting of the shoots, and successful reintroduction and establishment of the rooted plants in the forests of Palani hills in Southern India. A preliminary experiment on the culture of young nodal explants (1.0-1.5 cm) from field-grown plants in different basal nutrient media revealed high frequency bud beak in Schenk and Hildebrandt (SH) medium followed by Woody Plant medium and Murashige Skoog medium in 4 weeks. Subsequent culture of both shoot tips (0.5-0.8 cm) and nodes for 6 weeks in SH medium supplemented with various concentration of cytokinins resulted in optimal formation of 3.6 shoots in 80% of the nodes against 1.35 shoots in 75% of the shoot tips in the presence of 2.0 and 1.0 mg/l benzyl adenine (BA), respectively. Invariably, the resident apical and axillary meristems grew into a single shoot (1.0-1.8 cm) in 1-2 weeks followed by the characteristic differentiation of additional buds from its base. A synergistic combination of 1.0 mg/l BA and 0.02 mg/l indole-3-acetic acid (IAA) induced the maximum number (5.9) of axillary shoot formation which were relatively high (75%) when the explant was collected during May-June and the fifth nodes from the top of the growing shoots were used. Repeated subculture of the nodes from shoot cultures at 5-6 week intervals in medium supplemented with reduced concentrations of the growth regulators (0.5 mg/l BA, 0.01 mg/l IAA) through at least 10 passages enabled consistent production of 6-7 shoots per node at 92% success rate without loss of vigour, growth and morphological abnormalities. Shoots of 3-6 cm were rooted in vitro in the presence of 1.0 mg/l indole-3-butyric acid (IBA) and hardening in the mist house at 76-78% and these rooted plants established in a potting medium of river sand and top soil (1:1) under constant mist irrigation. The plants reared in the nursery for 5-8 weeks were successfully reintroduced into the natural forest segment of the Institute's campus (MSL 200 m) at Palode and Vattakanal shola forests of Palani hills (MSL 2500 m) with establishment frequencies of 78.8 and 90.6, respectively, after 18 months. Growth characteristics of the plants (height, number of leaves and branches) reintroduced into native habitat at Vattakanal were better than those at Palode. Genetic fidelity of the 12-month old plants established in the nursery/field was confirmed by analysis of amplified products of genomic DNA obtained with random primers.

ACCLIMATIZATION STUDIES IN STEVIA (STEVIA REBAUDIANA BERT.)

Considerable efforts have been directed to optimize the conditions for in vitro stages of micropropagation, but the process of acclimatization of micropropagated plants remains to be not meticulously studied and continues to be a major bottleneck in the micropropagation of many plants. Plantlets were developed within the culture vessels under low level of light, aseptic conditions, on a medium containing ample sugar and nutrients to allow for heterotrophic growth and in an atmosphere with high level of humidity. These contribute a culture-induced phenotype that cannot survive the environmental conditions when directly placed in a greenhouse or field. The physiological and anatomical characteristics of micropropagated plantlets necessitate that they should be gradually acclimatized to the environment of the greenhouse or field. Acclimatization continues to be a major hurdle in the micropropagation of stevia too with lower success reported. In the present study, different hardening media were used under different environments to improve post acclimatization survival. With sand as the hardening media, mist house had higher survival rates than greenhouse. Sand did not retain much water in greenhouse to ensure proper growth and survival compared with mist house with frequent misting. In all other cases, the survival was found to be better in greenhouse (27.89%) compared with mist house (23.11%). With frequent misting, water logging occurred around the plantlets, and they started to rot which lead to reduction in survival. This was absent in sand due to its high porosity and rate of percolation. Inclusion of cocopeat 75% or above in the hardening media brought a dramatic increase in the survival percentage. The survival of the micropropagated plantlets rose above 50% with the addition of 75% cocopeat to the hardening media. The survival percent recorded with cocopeat under greenhouse was around 75.50% that was almost 60 times higher than the lowest survival recorded with 100% sand under greenhouse (1.50%). On an average, sand as a hardening media, gave only 1.35% survival.

In vitro asymbiotic seed germination and seedling growth of Ansellia africana Lindl.

The effects of sodium hypochlorite treatment and growth medium on in vitro seed germination and seedling growth of the leopard orchid ( Ansellia africana Lindl.) were investigated. Forty minutes treatment of seeds with 1.75% sodium hypochlorite stimulated germination (70.6%) and seedling development when measured at Stage 5 (emergence of first leaf) on P668 medium after 8 weeks of culture. The growth medium played a significant role in determining the germination response of A. africana seeds. Dark pretreatment of seed cultures significantly enhanced the germination percentage and the growth of rhizoids on the protocorms. Leaf growth in terms of length was substantially enhanced on P668 medium. It was significantly reduced in modified Knudson C medium after 16 weeks of culture. Seedlings developed on P668 medium showed a significantly better growth performance in relation to leaf length, leaf number, root length, fresh and dry weights per plant in vermiculite after 12 weeks of ex vitro growth in a mist house with 90% relative humidity. Further studies developing a suitable method for in vitro symbiotic germination could assist in reintroduction of this threatened orchid species into the wild.

Micropropagation of Searsia dentata

A method for in vitro regeneration of Searsia dentata from nodal and shoot tip explants derived from mature trees is outlined. Nodal explants produced multiple shoots from the axis when cultured on Murashige and Skoog (MS) medium containing 3% sucrose supplemented with 0, 5, 7.5, 10, or 12.5 μM N6-benzyladenine (BA). An average of 5.3 shoots was obtained from nodal explants on 10 μM BA. For shoot tip explants, however, supplementation of α-naphthaleneacetic acid (NAA) with BA favored a caulogenic response. A maximum of 6.1 shoots were produced per shoot tip explant on MS containing 7.5 μM BA plus 5.0 μM NAA. The in vitro-regenerated shoots produced roots when transferred to full-strength MS medium containing 3% sucrose and 10 μM indole-3-butyric acid (IBA). The developed plantlets were transferred initially to a mist house. After an initial acclimatization period of 3–4 mo, plantlets were shifted to the greenhouse where they thrived for 9 mo. The standardized protocol for mass propagation of S. dentata should eliminate the dependence on natural stands of plants for traditional medicinal purposes, and will also serve as a means of conservation as the species is heavily overexploited.

English

Eight strains of Bradyrhizobium japonicum were isolated from the root nodules of soybean cultivar PK 472 collected from Adaptive Trial Centre, Bundi, India. All the isolates were authenticated through plant assay test in germination pouches. Growth of the isolated strains on different pH levels was observed and three strains namely Bj-3, Bj-5 and Bj-8 were found to be able to grow up to pH 8.5. Symbiotic efficiency of these isolates was determined under the mist house conditions in the sterilized sandy soil (pH 8.4). Maximum and minimum nodulation and vegetative growth were observed in Bj-3 and Bj-2 inoculated soybean plants, respectively. Three pH tolerant strains could also pose better results in the efficiency determination experiment. Considerable variability in terms of the symbiotic effectiveness was found in the tested strains.

ROOTING AND ACCLIMATIZATION OF MICROPROPAGATED TROPICAL FRUIT TREES AND MEDICINAL PLANTS

The in vitro shoots of Carica papaya 'Eksotika' cultured in MS medium containing a combination of 0.5 mg l -1 BA and 6 mg l -1 to 10 mg l -1 IBA produced roots which were thick and stumpy accompanied by callus tissue at the basal end. Papaya microcuttings dipped in 2.5 g l -1 IBA and then cultured in medium of distilled water plus 10 g 1 agar (Difco-bacto) produced normal shoots without any callus forming at the basal end but only 68% of the microcuttings rooted within three weeks. Only 30% of the rooted plantlets survived when transferred to the soil. However 69.4% of the papaya microcuttings rooted extra vitrum in Jiffy-7® and 95.5% of these rooted plantlets survived when transplanting. All of the Musa sapientum in vitro shoots produced roots within two weeks when cultured on MS basic medium and 93% of the rooted plantlets survived after being acclimatized in intermittent mist house for 10-14 days. Rooting of Cymbopogon nardus could also be induced directly by culturing the in vitro shoots into basic MS medium within two weeks. One hundred percent of the in vitro plantlets planted in Jiffy-7® survived after placing in an incubator with a temperature of 25±2°C for three weeks followed by transferring to the plant house. MS medium supplemented with 6 mg l -1 IBA induced 80% of the in vitro Orthosiphon stamineus shoots to root forming complete plantlets. All the rooted plantlets survived after acclimatization in the incubator with a temperature of 25±2°C for two weeks. Rooting of Typhonium flagelliforme were induced by planting extra vitrum the in vitro shoots in 1 sand: 1 coconut husks (by volume) mixture. Only 25% of the in vitro shoots of T. flagelliforme rooted and survived after acclimatization in intermittent mist house for two weeks. With all the leaves removed, 95-100% of the in vitro shoots of T. flagelliforme produced roots and survived under the same condition.

Propagation of Pennisetum setaceum ‘Rubrum’ from Cuttings

Abstract The effect of rooting medium, indole-3-butyric acid (IBA), and node position were studied on rooting of tender purple fountaingrass, Pennisetum setaceum ‘Rubrum’. Three-hundred single-node culm cuttings were placed in a mist house in sand, peat, vermiculite, perlite, or a peat:perlite (1:1 by vol) mix. Within 42 days, 78% of the cuttings rooted, with the highest number of cuttings rooting in the peat or perlite media and the lowest number in the vermiculite medium. Cuttings from the most proximal node exhibited a significantly higher percentage of rooting and produced more roots than cuttings from more distal nodes. Rooting medium and nodal position of cutting interacted to affect root dry weight. IBA had no significant effect on rooting. Propagation of purple fountaingrass from culm cuttings offers an economically-attractive alternative to the conventional method of crown division.

In vitro culture and establishment of Anthurium parvispathum

Sterilized seed of Anthurium parvispathum were germinated and used as explant source. The decontaminated germinated seedlings were excised and placed onto a multiplication medium containing 2 mg l− 1 BA and 0.2 mg l− 1 NAA. On average a 4-fold increase in the number of shoots produced was achieved on this medium. The shoots were subsequently placed on an elongation medium containing 20 mg l− 1 kinetin. The shoots elongated to a length of 2.3 cm. Shoot multiplication also occurred. These were then placed onto different rooting media. The optimal rooting medium contained 0.25 mg l− 1 IBA which produced 3.6 roots per plantlet with a 94% success rate. It took 24 weeks to progress from seed to rooted plantlets ready for acclimatization. Micropropagated plants were soaked in 0.2% Benlate to reduce fungal contamination and then placed into trays containing peat, bark and soil (1:1:1) for acclimatization. The trays were kept in a mist house for 4 weeks with overhead misting and bottom heat of 30 °C. They were then transferred to a green house where they were watered twice a week and sprayed with Kelpak (a seaweed concentrate) at a 1:500 dilution every second week. There was only a 2% loss during acclimatization and over the subsequent three months of growth.

In vitro propagation of some Cyrtanthus species

Shoots and roots were initiated on bulb explants of Cyrtanthus brachyscyphus , C . elatus , C . falcatus , C . guthrieae , and C . mackenii var. mackenii . C . breviflorus produced small amounts of wound callus only. The species differed in their response to the different levels of plant growth regulators used. In general shoot formation was most favourable with high concentrations BA (2 mgl − 1 ) and lower concentrations NAA (1 mgl − 1 ). Best root formation was obtained with low BA and NAA (0–0.5 mgl − 1 ) concentrations. Cyrtanthus brachyscyphus was the most prolific shoot producer, with a 3-fold increase at every sub-culture. C . elatus , C . guthrieae , and C . mackenii var. mackenii were less vigorous and on average showed a 1.5-fold increase at every sub-culture. C . falcatus produced a low number of shoots from the explants and this did not increase with subsequent sub-cultures. Rooted plantlets were successfully acclimatized in vermiculite in a mist house (100% survival).

In vitro culture of Gladiolus carneus

Sterile-grown seedlings of Gladiolus carneus were excised in root, hypocotyl and leaf expiants. The expiants were placed on Murashige and Skoog medium supplemented with 100 mg − 1 myoinositol, 3% sucrose and 1 mg l − 1 NAA and BA respectively, solidified with 0.8% agar. Multiple shoots occurred from the hypocotyl sections. The shoots readily formed corms. The cormlets were rooted on hormone-free MS medium. Plantlets were successfully hardened off in a sand:soil 2:1 mix in the mist house for four weeks, whereafter they were transferred to a greenhouse.

Micropropagation of Babiana Spp.

Seeds of six species of Babiana were germinated aseptically. Once germinated the seedlings were allowed to grow until 6 cm in height and were then cut into pieces giving root, hypocotyl and leaf expiants. These were placed on Murashige and Skoog medium containing 4.4 μM 6-benzylaminopurine and 5.3 μM 1-naphthaleneacetic acid. After 2–3 weeks multiple shoot formation occurred on the hypocotyl portion. Maximum shooting occurred on medium containing 8.8 and 22.1 μM 6-benzylaminopurine and no 1-naphthaleneacetic acid. Optimal rooting occurred on a hormone free medium. After rooting, plants from all species were hardened off in vermiculite or Genulite in a mist house for 3–4 weeks. Plants were then transplanted into a soil and sand mix (1:2 ratio). The plants were successfully hardened-off and flowered 6–8 months later