Single-node Culture Research Articles

Micropropagation of Lisianthus [Eustoma grandiflorum (Raf.) Shinn.] Leaf Explants and Single Nodes in Response to Plant Growth Regulators

Lisianthus has long-lasting, white, blue-purple, velvet or pink flowers. Varieties that can be used in pots, outdoors, or cut flowers are widely used and it is essential to develop new varieties to increase their commercial value. Breeding studies with classical methods can take a long time in lisianthus. Techniques to create variation in these plants and facilitate their reproduction always contribute to shortening the breeding period and one of the best known of these methods is plant tissue culture. This study aimed to investigate the micropropagation possibilities in lisianthus and examine the effects of auxin and cytokinin applications and doses on shoot reproduction. Supplemented with 10 different plant growth regulators in Murashige and Skoog (MS) medium, leaf explants from lisianthus plants that were germinated under in vitro conditions (seedlings) were cultivated. 6-benzyl aminopurine (BAP) was utilized in 5 doses (0.5, 1.0, 2.0, 3.0, and 4.0 mg L-1) alone in the first 5 groups. Naphthalene acetic acid (NAA) was also added to the BAP mediums at a concentration of 0.5 mg L-1. Shoot formation, shoot lengths, the number of shoots longer and shorter than 1 cm, and regeneration of new shoots after subcultures were determined. According to the results, MS mediums containing 3.0 and 4.0 mg L-1 BAP could be good starting mediums for regenerating new lisianthus shoots from leaf explants. These treatments got the highest values both in shoot formation and the number of shoots longer than 1 cm. It has been determined that 3.0 mg L-1 BAP alone can be used for both organogenesis from leaf explants and shoot proliferation in single node culture. In vitro shoots of the lisianthus species were easily rooted and both PGR-free MS or ½ MS mediums. Lisianthus shoots propagated under in vitro conditions can be rooted under ex vitro conditions both in the float hydroculture and in the soil mixture. The acclimatization stage was optimized also successfully.

Establishment and clonal propagation of Lippia dulcis Trevir. through in vitro single node cultures

Lippia dulcis Trevir. (Verbenaceae) is a species with medicinal potential that is used as a tranquilizer and to control diabetes in the western region of Pará, Brazil. The objective of this study was to determine the establishment and clonal propagation of Lippia dulcis through single nodal segments grown in different concentrations of salts in a MS (Murashige and Skoog) medium (MS, MS/2, MS/4) and determine the best period for subculturing the species. Nodal segments cultivated in the MS medium showed a higher survival percentage and better development than those grown in more dilute media (MS/2, MS/4). The concentration of the salts affected the dry weight gain of some plant parts. Plantlets cultivated in the MS medium obtained better leaf dry weight and total leaf area than plantlets cultivated in half-strength MS. The half-strength MS medium proved to be most effective for root length growth and root dry weight gainin vitro. The growth curve showed that day 30 was the time for subculturing the species. Starting with only one nodal segment, 3125 plantlets were obtained after sixth months of cultivation at a multiplication rate of five. The plantlets of Lippia dulcis were removed from the flasks and acclimatized with success in a greenhouse.Index terms: Growth analysis; micropropagation; shoot proliferation; nodal segment.

Micropropagation of Mountain Mulberry (Morus bombycis Koidz.) 'Kenmochi' on Cytokinin-Free Medium.

The aim of the study was to compare two methods of micropropagation of mulberry: single-node culture (“SNC”), and axillary-branching (“AxB”). The experiments were carried out on in vitro cultures for 6 successive passages. The “AxB” cultures were propagated on modified MS medium (+ 25% Ca2+ and Mg2+), supplemented with WPM vitamins, sucrose (30 g L−1), and BA (1.5 mg l-1). The “SNC” cultures were grown on cytokinin-free 1/2 MS (macro- and micronutrients) medium supplemented with WPM vitamins, IBA (0.05 mg l-1), and sucrose (15 g l-1). Both media (pH 5.8) were solidified with agar (7.0 g l-1). Initiation of in vitro cultures from explants taken from adult trees and young, potted plants was feasible on both media. Cultures were established from about 1 cm long nodal explants. Generally “SNC” cultures formed one well rooted, significantly longer axillary shoot with bigger leaves than “AxB” cultures, which developed significantly more shoots and big callus at the explant base. All shoots collected from “SNC” and “AxB” cultures rooted in vivo in peat mixture and developed into similar plantlets. The single-node method based on application of cytokinin-free medium is a good alternative for the axillary-branching method for micropropagation of mountain mulberry (Morus bombycis) ‘Kenmochi”’.

Einfluss von BAP-Konzentrationen und Nährmedienzusammensetzung auf die in vitro Revitalisierung bei den Weinreben-Sorten Öküzgözü’ und ‘Boğazkere’ (Vitis vinifera L.)

This study has been conducted with the aim to determine the type of nutrient medium that can be used in micropropagation studies for ‘Okuzgozu’ and ‘Bogazkere’ and to specify BAP concentrations. In the study where ejectors with a length of 0.7–0.8 cm that are obtained with single-node culture are used, it was focused on four different nutrient media such as MS, DKW, QL and WPM and on six different concentrations such as 0.2–0.4–0.6–0.8–1.0–1.5 mg l−1 BAP. Single-node suspension explants which will be used in initiating the culture, are taken into culture in MS nutrient medium and the nutrient medium is supported with 30 g l−1 sucrose, 6 g l−1 agar and 1 mg l−1 BAP. In the trial environment, parameters such as number of shoots, shoot length (cm), number of nodes and callus ratio have been investigated. For both grape varieties, the best outcome was obtained with MS nutrient medium with respect to number of shoots, shoot length, and number of nodes. These values were found as 4.66, 1.24 and 6.39 for ‘Okuzgozu’ variety respectively, whereas they are determined as 6.28, 1.15 and 6.81 for ‘Bogazkere’ variety respectively. In both grape varieties in DKW nutrient medium, starting from the 2nd week of culture, obscuration began to appear on the shoots and after this stage no other development has taken place.

Determinación del nivel de tolerancia a salinidad en condiciones <em>in vitro</em> de plantas de tomate de árbol (<em>Solanum betaceum</em>) provenientes de distintas localidades de la Sierra ecuatoriana

La salinidad de los suelos constituye una de las causas más importantes para la disminución de la productividad en cultivos agrícolas. Gran parte de los suelos de la Sierra ecuatoriana son considerados salinos, tanto por su naturaleza piroclástica como por la erosión y el mal uso del agua de riego. El tomate de árbol (Solanum betaceum) crece en la región interandina y en muchos casos se ve sometido al estrés salino. El objetivo del presente estudio fue determinar el grado de tolerancia del tomate de árbol a cloruro de sodio (NaCl) en cultivo in vitro. Se utilizó plantas de tomate de árbol provenientes de siete u ocho localidades dependiendo del ensayo realizado. Se evaluó la germinación de semillas, el desarrollo de plántulas a partir de nudo simple y el crecimiento de plántulas cultivadas in vitro en diferentes concentraciones de NaCl. En los ensayos de germinación de semillas se obtuvo un promedio de 62% de germinación para siete localidades analizadas en una concentración de 50mM de NaCl, y un promedio de 57% en una concentración de 75mM. En cuanto al cultivo de tomate de árbol a partir de nudo simple, se evaluó plantas de siete localidades de las cuales todas, con excepción de Quero, crecieron hasta en una concentración de 100mM de NaCl. Finalmente, en el ensayo para determinar el límite de tolerancia a NaCl en el crecimiento de plántulas, se observó diferentes respuestas al estrés salino en individuos de tomate de árbol de ocho localidades estudiadas, siendo las plántulas de Chaltura las que toleraron hasta 175 mM de NaCl. Estos resultados permiten afirmar que hubo variaciones en el grado de tolerancia a salinidad entre plantas de tomate de árbol de las diferentes localidades. La mayor tolerancia se encontró en las plantas provenientes de Chaltura, y la menor tolerancia en plantas provenientes de Quero. La información obtenida en este estudio es la base para posteriores análisis del transcriptoma donde se pueda identificar genes involucrados en la respuesta a la tolerancia a la salinidad en este interesante cultivo andino.

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.

Cost effective slow growth in vitro conservation of potato (Solanum tuberosum L.) using table sugar as an alternative carbon source

The potential of table sugar as an alternative low cost carbon source for potato in vitroconservation media at normal propagation temperature was investigated using a two factor experiment in a randomized complete block design. Three types of carbon sources: (1) white sugar, (2) brown sugar and (3) laboratory grade sucrose, were investigated using three varieties (Arka, Dutch Robijn and Tigoni). Plantlet survival after 18 months of conservation was similar for all the three carbon sources evaluated for cultivars Arka and Dutch Robijn but was significantly lower for cultivar Tigoni (65.0 to 68.8%). Cultivar Tigoni had the poorest condition of plantlets for all media evaluated (1.8 to 1.9). The number of usable single node cuttings/culture ranged from 5.3 to 11.2. Cultivars Tigoni and Dutch Robijn gave the highest number of usable single node cuttings/culture for each of the three media evaluated. Conservation costs were reduced by 96.4% when both white and brown sugar were used as the source of carbon instead of laboratory grade sucrose. All plantlets that survived the 18-month conservation period had 100% viability irrespective of the type of carbon source used in the conservation media. Table sugar may therefore be used as a cheaper substitute for laboratory grade sucrose in potato conservation media. Key words: Table sugar, carbon source, in vitro conservation, normal propagation temperature, potato, slow growth, single node cultures.

Micropropagation of the apricot (Prunus armeniaca L.) cv. Hacıhaliloğlu by means of single node culture

Hacıhaliloğlu is one of the most widely cultivated apricot (Prunus armeniaca L.) cultivars in Turkey. It produces fruit of excellent quality and is consumed both fresh and dried. In this study several factors affecting in vitro culture, rapid proliferation, rooting, and acclimatization of the apricot cultivar Hacıhaliloğlu were examined. During this study significant differences in harvesting dates were observed, with the month of May being noted as the best time to examine the in vitro culture of Hacıhaliloğlu. The effects of the cytokinin 6-benzylaminopurine (BA), kinetin (Kin), and thidiazuron (TDZ) were evaluated individually, and different concentrations of BA were also tested. BA levels were tested for their effects on shoot proliferation and 1.0 mg L^{-1} of BA was the most suitable dose for promoting shoot multiplication cultures. The greatest number of shoots (3.42 ± 0.19) was obtained using 2.0 mg L^{-1} of BA; this was significantly greater than that of the control, but there was no significant difference in the mean obtained with 1 mg L^{-1} of BA. The effect of a carbon source on shoot proliferation was studied by using glucose, sucrose, fructose, and lactose at the 3.0% concentration. Sucrose resulted in better shoot proliferation than the other sugar varieties tested. The best rooting percentage was obtained on Murashige and Skoog (MS) medium supplemented with 2.0 mg L^{-1} of indole-3-butyric acid (IBA). The regenerated plants were successfully transferred to soil. Herein a simple and effective method is reported for the micropropagation of the apricot cv. Hacıhaliloğlu from adult plants.

The effect of plant growth regulators, cultivars and substrate combination on production of virus free potato minitubers

Nowadays large amounts of potato seed in the world is produced by in vitro virus free minitubers. Therefore, evaluation of commercial varieties in production of virus free potato minitubers is critical. In this study virus free plantlets of 4 potato cultivars of Agria, Marfona, Sante and Burren were achieved with meristem culture method. The meristem-derived plantlets which had optimum growth were transferred to the MS-medium without any growth regulators to stimulate the same vigor of the plantlets. Then DAS-ELISA test was conducted and the effects of different level of
2 growth regulators either singly or in combination were evaluated on single node culture of potato. In all cultivars the best and most economical medium for single node culture was MS medium without growth regulators. Afterwards virus free plantlets were selected and transferred to the green house. The effects of substrate combination including 4 planting bed in minituber production were evaluated, after 90 days, numbers and diameters of minitubers for each cultivar were counted and recorded. Marfona had the greatest number of minitubers and Agria had the lowest. There are no any significant differences in minituber diameter between the cultivars. A mixture of peat moss and sand in the ratio of 1:1 was proved to be the best for minituber production.

감자절간배양에서 마디위치 및 잎의 부착유무에 따른 소괴경형성의 차이

감자의 마디배양에서 이식하는 마디의 위치에 따라 기내 소괴경 형성에 미치는 차이와 배양하는 마디조직체에서의 잎의 유무가 기내소괴경의 형성에 미치는 온도, 일장 및 CCC의 영향을 검토하였다. 마디의 위치에 따른 감자의 생육에 있어서는 초장, 생체 중, 직경, 마디수는 물론 괴경형성에 있어서도 정단부위가 하부부위보다 양호하였다. 액아의 부위에서 형성된 소괴경의 직경에 있어서는 정단부를 포함한 모든 처리구에서 큰 차이를 확인할 수 없었으나 괴경의 생체중은 정단부위의 액아에서 형성된 괴경에서 높은 수치를 보였고 다른 마디에서는 개체간의 큰 차이가 없었다. 잎의 부착 유무와 온도와 일장에 따른 괴경형성에 있어서는 잎이 부착된 마디를 <TEX>$20^{\circ}C$</TEX>의 단일조건에서 배양할 때 양호하였으나, 고온에서는 괴경형성이 일장에 관계없이 저조하였다. 잎이 부착되지 않은 마디에서의 괴경형성은 저온과 고온 모두 단일조건에서 양호하였다. CCC처리에 의한 효과에 따른 감자의 생육에 있어서 초장, 생체중, 직경, 마디수는 CCC를 첨가한 배지에서 양호하였고 특히 일장 처리가 길어질수록 양호한 결과를 보였다. 괴경형성을 촉진시키는데 일장이 짧을수록 양호하였고, CCC를 첨가한 배지에서 양호한 효과를 보였다. Single-node stem pieces ca. 1 cm in length containing a axillary bud were obtained from in vitro plants of potato (Solanum tuberosum L.). The influences by a position of the node and the existence of a leaf at the node were observed in the single-node culture on the 8% sucrose MS medium. The effect of CCC was also investigated for the microtuberization. The apical part node was excellent in the tuberization not to mention shoot length, fresh weight, diameter, the number of node on the in vitro culture of a single-node than the lower part. The differences in the diameter of a tuber formed in the part of the axillary bud on all treatments including the cultivation of the apical part node were not recognized. However, the fresh weight of the tuber showed high value in the tuber formed at the axillary bud of shoot apex part. At 20 days after cultivation, tuberization was promoted in the new stolen that developed from the bud of node with a leaf under SD condition of 8 hours at <TEX>$20^{\circ}C$</TEX>. The tuberization from axillary bud of the single-node without leaf was inhibited at high temperature of <TEX>$28^{\circ}C$</TEX> regardless of daylength. Whereas, tuberization at <TEX>$20^{\circ}C$</TEX> and <TEX>$28^{\circ}C$</TEX> was similar without the difference under SD condition but the tuber formation ratio were low. CCC 500 mg/L promoted tuberization and the effect was also showed even under LD condition at <TEX>$28^{\circ}C$</TEX>. The inhibiton of tuberization under LD and high temperature condition could be solved by treatment with CCC.

고구마 정단분열조직 유래 식물체의 기내 증식에 미치는 배양조건의 영향

Sweet potato is a crop vegetatively propagated by vine cuttings, an ineffective method for maintaining pathogene-free stock plants. As an alternative method, single-node cultures of virus-free plantlets derived from apical meristem in sweet potato (cv. Yulmi) was examined. Effective pH range, sugar concentration and nodal order were investigated to establish an in vitro mass propagation system with high quality virus-free stock plantlets to farmhouse. Although the plantlets grew at wide range of pH, the most effective pH of the medium was 4.8 in single-node cultures. High sugar concentration of 60∼80 g/L resulted in increased growth response in shoot length, root length, number of node, leaf area and fresh and dry weight of shoot and root, whereas reducing sugar contents below 6% was showed reduced growth response. The first node including meristem tip was the best for the rapid growth of plantlets and the other nodes also showed a very similar growth response. Uniform plantlet can be obtained massively at the same time by culture of single node except for the first node including meristem tip. In conclusion, the most effective pH range and sugar concentration of medium for the growth of plantlets via single-node cultures was 4.8, 60∼80 g/L respectively. The first node was the best for the rapid propagation of plantlets in nodal order.

Effect of Different Media Additives on In vitro Culture of Potato (Solanum tuberosum)

Effect of different media additives on in vitro culture of three potato varieties viz.Lalpakri, Cardinal and BARI TPS-1 was investigated using single nodes as explant. The single nodes were collected from microplants of tuber sprouts that was preincubated under dark. Survivability of the cultures was statistically insignificant in respect of the media used, but the highest percentage of cultures survived on MS medium supplimented with 60 mg L-1 inositol. The time taken to initiate new shoots was minimum on MS medium supplemented with 1 mg L-1 Benzyle Amino Purine (BAP). The number of shoot was maximum on a medium containing MS + 0.25 mg L-1 Gibberellic Acid (GA3) and 2 mg L-1 Calcium Pantothenate. The maximum number of roots was obtained on medium containing MS + 0.03 mg L-1 Naphthalene Acetic Acid (NAA). Among the varieties tested, BARI TPS-1 performed better in respect of single node culture. The interaction between different potato varieties and media compositions was significant for different characters of in vitro grown plantlets.

Acmella oppositifolia micropropagation by single-node culture

Acmella oppositifolia plantlet formation was achieved by subculturing single-node explants on Murashige and Skoog medium without growth regulators. The explants from 1-month-old in vitro plantlets produced shoots over a 7-day culture period. From these in vitro cultured nodes readily rooted shoots elongated on auxin-free MS medium. Plants produced were easily acclimatized and subsequently flowered in a greenhouse. This species is of medicinal value in tropical America from Mexico to Colombia.

Modified single node culture method—a new micropropagation method for chickpea

Chickpea was micropropagated by axillary shoot proliferation (ASP) and modified single node culture (MSNC) methods. Maximum propagule proliferation occurred on Murashige and Skoog (MS) medium enriched with 1–10 µM N6-benzyladenine and 0.01 µM α-naphthaleneacetic acid. The propagules were rooted on MS medium containing 1 µM 3-indolebutyric acid and B5 vitamins. Regenerated plants were fertile and phenotypically similar to control plants grown from seed. The MSNC method was four times more efficient than the ASP method in terms of the number of plants produced per explant.

トルコギキョウの組織培養による大量増殖

トルコギキョウの組織培養による大量増殖

COMPARATIVE ULTRASTRUCTURES OF VITREOUS AND NON-VITREOUS SWEET POTATO (IPOMOEA BATATAS L.)LAM. PLANTS GROWN IN VITRO

Vitrification, a physiological disorder characteristic of in vitro grown plants, was observed in single-node cultures of sweet potato in mannitol-enriched medium during their second year of storage. Vitrified or vitreous sweet potato plantlets were watersoaked, translucent or glassy in appearance, with thick, swollen, leaves and stems, stunted shoot growth and poor root growth. These plantlets were not able to regenerate normal plants when transferred into fresh regeneration medium nor were they able to survive outside culture conditions. Electron microscopy revealed changes in the ultrastructures of vitrified sweet potato plantlets. Vitrified plants had defective stomatal complex, starch grain-filled chloroplasts, disrupted cell wall, big air spaces (lacunae), high frequency of cell membrane separation from the cell wall, nuclear disintegration, and cytoplasmic disorganization. These changes in the internal structures of vitrified plants were reflected in their abnormal morphology and physiology.

COMPARATIVE ULTRASTRUCTURES OF VITREOUS AND NON-VITREOUS SWEET POTATO (IPOMOEA BATATAS L.)LAM. PLANTS GROWN IN VITRO

Vitrification, a physiological disorder characteristic of in vitro grown plants, was observed in single-node cultures of sweet potato in mannitol-enriched medium during their second year of storage. Vitrified or vitreous sweet potato plantlets were watersoaked, translucent or glassy in appearance, with thick, swollen, leaves and stems, stunted shoot growth and poor root growth. These plantlets were not able to regenerate normal plants when transferred into fresh regeneration medium nor were they able to survive outside culture conditions. Electron microscopy revealed changes in the ultrastructures of vitrified sweet potato plantlets. Vitrified plants had defective stomatal complex, starch grain-filled chloroplasts, disrupted cell wall, big air spaces (lacunae), high frequency of cell membrane separation from the cell wall, nuclear disintegration, and cytoplasmic disorganization. These changes in the internal structures of vitrified plants were reflected in their abnormal morphology and physiology.

In vitro propagation of the alkaloid producing plant Datura insignis Barb. Rodr.

A method is presented for the in vitro propagation of Datura insignis Barb. Rodr. Nodal explants were cultured on Murashige and Skoog medium supplemented either with BA alone or in combination with 2,4-D or IAA. Shoot multiplication and elongation were obtained in various growth regulator concentrations. Best results were obtained in a medium with 1.0 mgl-1 of BA. Individual shoots were excised and transfered to growth regulator-free medium for rooting. Additional multiplication was obtained by single-node culture using explants from these in vitro rooted shoots. Rooted plantlets were successfully grown in soil.

* VEGETATIVE PROPAGATION OF SYRINGA VULGARIS L. IN VITRO

Excised shoot tips from adult Syringa vulgaris L. plants were rejuvenated by repeated subculturing in vitro. The number of subcultures required to rejuvenate the shoots was strongly dependent on the age and genotype of the plant material. Three rootstocks (K8, A2 and A3) and 5 cultivars (Mademoiselle Marie Legray, Madame Florent Stepman, Marechal Foch, Hugo Koster and Herman Eilers) were studied in vitro. The basic culture medium for isolation, subculture and shoot elongation contained: Murashige and Skoog (MS) macro-salts 1.5 strength, MS micro-salts (except Fe), NaFeEDTA 50 mg/1, saccharose 3.5°2iP 0.8 mg/1, Difco Bacto-agar 0.8°pH 6.0 and distilled water. The cultures were incubated in a growth room at 21°C and a day/night schedule of 16 h fluorescent light/8 h darkness. After rejuvenation, cloning was realized by repeated single-node culture. The application of a cytokinin and low irradiance (4–5 W m-2) induced stem elongation from pre-formed buds in the axils of the leaves. Zeatin(riboside) was most effective for stem elongation, followed by 2iP. The cytokinins, BA and kinetin, were not very effective in inducing stem elongation. Stem elongation was dependent on the genotype used. High cytokinin levels (2–5 mg/1) induced axillary branching but hardly any stem elongation and the leaves were strongly curled. Single-node culture and stem elongation in a medium containing 1.0 mg/1 2iP was definitely the best method of propagating lilac in vitro. Shoots were easy to root on a cytokinin-free medium with auxin or on an artificial substrate (rock wool) after an auxin dip. Acclimatization of in vitro rooted shoots to greenhouse conditions was successful when at low irradiance and high relative humidity for the first 2 weeks after transfer.

* IN VITRO CULTURE OF HIGHER PLANTS AS A TOOL IN THE PROPAGATION OF HORTICULTURAL CROPS

In vitro culture of higher plants is the culture, under sterile conditions, of plants, seeds, embryos, organs, explants, tissues, cells and protoplasts on nutrient media. This type of culture has shown spectacular development since 1975, resulting in the production and regeneration of viable individuals of many plant species. In addition, since 1980 there has been an explosion in the development of genetic manipulation and biotechnology. Various types of cultures and their applications are discussed: embryo culture, orchid seed culture, meristem culture, cloning of crops (single-node culture, meristem culture, axillary branching, adventitious shoot formation, callus culture, single cell and protoplast culture), anther and microspore culture, culture of ovules and excised flowers, and protoplast culture. Vegetative propagation in vitro (also called micropropagation) will be dealt with in greater detail since it has yielded results which are of enormous importance in horticulture. A recent survey in the Netherlands has shown that the number of commercial tissue culture laboratories in this country has increased from 28 in 1983 to 50 in 1986; the total number of plants cloned in vitro increasing from 21 to 43 million over the same period. The widespread use of in vitro cloning is an indication of its many advantages. However, there are also disadvantages. The criteria desired for the success of in vitro cloning can be itemized as follows: no induction of mutations; the starting material must be disease-free, or disease-free material must be produced by meristem culture; the transfer from test tube or container to soil should not be too difficult; the regeneration ability should not be lost; the technique should be economically viable and not too complicated (synthetic seeds!), otherwise it will be rejected. Woody species (trees and shrubs) are far more difficult to clone in vitro than herbaceous plants; the reasons for the differences are given. In vegetative propagation and propagation by seeds, in vitro culture can also be a tool to: obtain disease-free plants; transport disease-free plant material; increase the genetic variation (by somaclonal variation and genetic manipulation); induce haploids; separate chimeras; obtain tetraploids; store plant material in gene banks where it can be kept disease-free under conditions which limit growth and development.