Induction,growth and direct rooting of adventitious shoots of Begonia x hiemalis

The efficiency of commercial micropropagation programs for Begonia x hiemalis depends on the production of large adventitious shoots for easy handling and on effective rooting and acclimatization procedures. Maximum induction of adventitious buds on petiole segments occurred in response to NAA (0.1 mg, l^-1) and BA (0.5 mg l^-1), but continued shoot growth was limited. With a lower concentration of BA (0.1 mg l^-1) fewer shoots were produced but shoot growth was enhanced. With a combined agar/liquid culture program the low BA (0.1 mg l^-1) medium produced 50 percent more shoots larger than 1 cm than did the high BA (0.5 mg l^-1) medium. In vitro rooted explants developed weak root systems and acclimatization losses occurred during adaptation to greenhouse conditions. Adventitious shoots treated with commercial rooting powder and placed directly in mist frames produced much stronger root systems and could be adapted to greenhouse conditions without loss. The elimination of the in vitro rooting stage also simplifies the micropropagation program.Contribution No. 743     

Micropropagation of <Emphasis Type="Italic">Theobroma cacao</Emphasis> L. using somatic embryo-derived plants

Summary A micropropagation protocol was developed using cacao somatic embryo-derived plant as a source for nodal and apical stem explants, and apical microcuttings. Microcuttings were efficiently rooted and developed into plantlets. Axillary meristems within the remaining decapitated plantlets subsequently developed and were used for production of additional microcuttings, with an average 2.4 growing shoots per decapitated stem. The remaining plantelts were maintained as microcutting stock plants. When nodal stem explants were cultured on thidiazuron medium, axillary buds proliferated and developed into shoots, which were excised and rooted. However, the efficiency of this method is lower than rooting of apical microcuttings harvested directly from stock plants. During root induction, short treatment with indole-3-butyric acid (IBA) increased the total percentage of rooted microcuttings up to 89%. Longer exposures to IBA increased the average number of roots per microcutting (from 1.7 to 5.2). Plant acclimatization after rooting was achieved with an average success of 87%. During several months of growth in the greenhouse, the micropropagated plants developed functional taproots. Currently, cocoa plants produced by this micropropagation method have been successfully acclimated to field conditions in Ivory Coast, Ghana, and Saint Lucia.     

Micropropagation of Ilex aquifolium L.

Summary Two procedures were tested for micropropagation of Ilex aquifolium (English holly), one in which shoots proliferated on solid medium and another one using liquid medium. Different growth regulator treatments and supports were analyzed for optimizing in vitro rooting, showing that indolebutyric acid and agar or cellulose plugs gave the best results. The surival percentage of successfully in vitro or ex vitro rooted plants did not differ significantly between the best treatments. However, the efficiency with ex vitro rooting was 80%, while for in vitro rooting, the final efficiency was 64%. The results show that a correct manipulation of Murashige's stage II of micropropagation and eliminating or decreasing stage III are useful tools to reduce the requirements of acclimatization.     

Rooting affects the photosystem II activity: in vitro and ex vitro studies on energy hybrid sorrel

Rumex tianschanicus × Rumex patientia is a high-biomass-yielding plant suitable for fuel and biogas production. The protocol of the hybrid sorrel micropropagation was used to study the changes in the photosystem II (PSII) activity as well as to analyse the ultrastructure of the chloroplasts. The lowest effective PSII quantum yield [Y(II)] and an apparent electron transport rate of PSII [ETR(II)] were observed for adventitious shoots that had been regenerated in vitro, before rooting. These fluorescence parameters were higher and similar for both the leaves of the same adventitious shoots that had been rooted under in vitro conditions and for the shoots that had been acclimated and grown in ex vitro conditions. The analysis indicated that the PSII activity strongly depends on the formation of properly functioning roots and that in vitro or ex vitro culture conditions are, at least to some degree, less important. TEM analysis revealed that chloroplasts from plants rooted in vitro were sufficiently mature and acclimatization processes have less impact on their development. This is the first report concerning the analysis of PSII activity and the ultrastructure of the chloroplasts at all of the stages of micropropagation, i.e. adventitious shoot formation in vitro, rooting in vitro and acclimation to ex vitro conditions. It strongly indicated that rooting under in vitro conditions, rather than the acclimation to ex vitro conditions, plays a key role in the development of a completely functional photosynthetic apparatus in hybrid sorrel.     

Using LED lighting in somatic embryogenesis and micropropagation of an elite sugarcane variety and its effect on redox metabolism during acclimatization

This study investigated the ability of a light emitting diode (LED) to induce somatic embryogenesis (SE), shoot multiplication, and rooting of sugarcane (RB98710). We also accessed the effects on acclimatization. MS medium was used for all stages and was supplemented with different concentrations of growth regulators according to the culture stage. The material was maintained in a growth room under fluorescent (FL) or LED (82?% red, 18?% blue) lighting after rooting plants were acclimatized. We conducted both biometric and biochemical analyses before and after acclimatization. The LED conditions favored the formation of callus; however, the FL was more efficient at plant regeneration. A histological analysis showed the formation of somatic embryos occurred through direct and indirect pathways. The plants obtained through SE and grown under LED had a higher multiplication rate over six subcultures. Shoots rooted in both light sources, but the number of shoots and the weight gain of the roots were higher under LED. The malondialdehyde (MDA) level did not differ among treatments. Our results indicate the SE induction phase should be conducted under FL and the remaining micropropagation process should be performed using LED. After acclimatization the plants grown under LED did not change the SOD and CAT activities during the first 5 days, which suggests there was no acclimatization impact. The H₂O₂ and MDA values observed do not suggest damage to membranes. There was better development, lower water loss, and higher survival rate in plants from in vitro culture under LED conditions when compared to FL.     

Micropropagation of mature <Emphasis Type="Italic">Crataegus aronia</Emphasis> L., a medicinal and ornamental plant with rootstock potential for pome fruit

The effects of culture media and cytokinin types on micropropagation of mature Crataegus aronia L. were investigated. Using single-axillary bud explants, the growth of cultures on MS, WPM, DKW and NRM containing 4.44 μM benzyladenine (BA) plus 0.05 μM indole-3-butyric acid (IBA), and on NRM containing thidiazuron, meta-Topolin (mT) or BA at 1.25, 2.5, 5.0 or 7.5 μM plus 0.05 μM IBA were compared. The culture medium had significant effects on shoot number and length. In comparison with MS, DKW and WPM, shoot production was greater on NRM (5.7 shoots per explant). Shoot production on MS, DKW and WPM (4.2, 4.2 and 4.1, respectively) were statistically similar to each other. Thidiazuron was detrimental to shoot formation and caused formation of rosette shoots and/or large callus to form on explants. In the presence of mT, only some of the explants developed into shoots. Benzyladenine was the only cytokinin that promoted both shoot proliferation and shoot elongation. Higher shoot numbers were obtained at 5.0 and 7.5 μM BA compared to lower concentrations of BA. Over 80% of microshoots rooted and rooted shoots were successfully acclimatized to ex vitro conditions.     

Micropropagation of <Emphasis Type="Italic">Juniperus phoenicea</Emphasis> from adult plant explants and analysis of ploidy stability using flow cytometry

We report here the successful micropropagation of adult Juniperus phoenicea L. with respective ploidy stability studies. Microcuttings with axillary buds were grown on five media supplemented with different growth regulator combinations. Best elongation rates were achieved on Driver and Kuniyuki (DKW) medium supplemented with kinetin alone or with naphthaleneacetic acid (NAA), while Rugini olive (OM) medium stimulated the development of new branches. Shoots growing on Murashige and Skoog (MS) medium browned and showed necrotic zones. Shoots of second to fourth subcultures usually had higher elongation rates than those of the first culture. For rooting assays, half strength DKW and OM media, different concentrations of growth regulators, auxin continuous exposure vs. dipping and the type of solid matrix were assessed. During rooting assays, two morphotypes were observed with one type having well developed internodes and the other showing hyperhydratation and no internode development. High rooting rates (40 %) were only obtained in the first morphotype shoots exposed for 5 min to 2.4 μM IBA and then transferred to OM medium without growth regulators. Plants were acclimatized in pots containing a mixture of peat and Perlite (3:2) in greenhouse with progressive reduction of relative humidity. A flow cytometric screening for major ploidy changes revealed no differences among the morphotypes and between them and the mother plant. Also the nuclear DNA content of this species was estimated for the first time using flow cytometry (2C = 24.71 pg).     

李砧木Marianna离体叶片再生体系优化研究

以李砧木‘Marianna’试管苗新梢顶端第1片叶为外植体,研究激素组合、基本培养基种类及外植体类型等对不定芽再生的影响。结果表明:1/2 MS基本培养基和WPM培养基再生率显著高于MS和SH培养基;叶片附带叶柄的外植体再生率和再生不定芽数显著高于叶柄和切除叶柄的叶片外植体;最佳再生培养基为1/2MS+2.0mg/L TDZ+0.1 mg/L IBA+0.25%琼脂+3.0%蔗糖,最高再生率和再生不定芽数分别为81.7%和7.46±1.38个;最佳生根培养基为1/2MS+0.5~1.0 mg/L IBA,能获得96.7%生根率、较高的生根数和根长。     

Micropropagation from inflorescence stems of the Spanish endemic plant Centaurea paui Loscos ex Willk. (Compositae)

Tissue culture techniques have been established as a useful approach for ex situ conservation of rare, endemic or threatened plant species. This report describes the micropropagation of Centaurea paui Loscos ex Willk (Compositae), an extremely endangered plant species endemic to the Valencia Community (eastern Spain), as a conservation measure which does not cause damage to the wild plants used as explant source. Inflorescence nodal segments of C. paui were selected as explants for in vitro establishment. The best rate of shoot proliferation was obtained on Murashige and Skoog (MS) mineral medium supplemented with 0.5 mg/l 6-benzyladenine or with 2 mg/l kinetin. Maximum shoot elongation was achieved without growth regulators, and the addition of cytokinins significantly decreased their size. In vitro rooting of shoots was difficult after 6 weeks on rooting media. The combination of 2 mg/l indole-3-acetic acid plus 2 mg/l indole-3-butyric acid on MS medium yielded the best results. In this medium, 40% of shoots rooted before 30 days of culture. About 70% of the rooted plants were successfully transferred to pots and acclimatized to ex vitro conditions. Received: 12 January 1998 / Revision received: 10 October 1998 / Accepted: 28 October 1998     

In vitro Clonal Propagation of Neem (Azadirachta indica)

The micropropagation of neem (Azadirachta indica) was accomplished by culture of buds from crown branches of a mature tree, basal-sprouts of another mature tree and a single juvenile plant. Cultures derived from these three different sources showed significant variation in in vitro response. In case of the crown and basal-sprout explants, addition of 12.5 μM PVP-40 in the establishment medium controlled leaching of phenol growth inhibitors. Phenolic leaching was not observed in juvenile explants. DKW medium (with 0.22 μM BA) was significantly better than MS for shoot proliferation. Shoot cultures of crown branch origin did not elongate and eventually died after the third subculture. In the presence of 4.9 μM IBA in half-strength DKW, 90% of the shoots and 100% of basal-sprout and seedling explant origin, formed roots. Plantlets from both explant types showed 90% survival after acclimatization.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Acer grandidentatum</Emphasis> Nutt.

Bigtooth maple (Acer grandidentatum) is a promising ornamental tree that is not widely used in managed landscapes. Tissue culture has not been used successfully to propagate this taxon. We cultured single- and double-node explants from greenhouse-grown, 2-y old seedlings of bigtooth maples, which are indigenous to New Mexico, Texas, and Utah, on Murashige–Skoog (MS), Linsmaier–Skoog (LS), Driver–Kuniyuki Walnut (DKW), and Woody Plant (WPM) tissue culture media. Media affected shoot proliferation (P = 0.0242) but the zone of explant origin (P = 0.7594) did not. After four 30-d subcultures, explants on DKW media and WPM media produced 3.6 and 3.5 shoots per explant, respectively. Sprouting rates were highest on DKW, making DKW the best overall media for shoot proliferation. Double-node microshoots were rooted in vitro on DKW containing indole acetic acid (IAA). Microshoots represented six genotypes from three locations within Texas and New Mexico. Rooting percentage increased up to 15% as IAA concentration increased (P = 0.0040). There was 100% survival of rooted microshoots in vented Phytatrays containing one perlite: one peat moss (v/v). We conclude that DKW can be used to proliferate microshoots, and IAA induces rooting in microshoots of bigtooth maple.     

In vitro propagation of<Emphasis Type="Italic"> Saussurea obvallata</Emphasis> (DC.) Edgew.—an endangered ethnoreligious medicinal herb of Himalaya

This is the first report of a micropropagation protocol for Saussurea obvallata (DC.) Edgew. (Asteraceae), a rare, threatened and near-endemic medicinal herb of the Indian Himalayan region. Multiple shoots were formed from epicotyle explants on Murashige and Skoog (MS) medium supplemented with 1.0 microM kinetin and 0.25 microM alpha-naphthaleneacetic acid. A maximum of five shoots were obtained from one explant in a 75-day culture period. The effect of subsequent subcultures on shoot formation was also studied. After 100% in vitro rooting was obtained in half-strength MS supplemented with 2.5 microM indole-3-butyric acid, the plantlets were transferred to ex vitro conditions. Following a 15-day in vitro rooting period and 12 days of ex vitro acclimatization, 66.7% of the plantlets had established in the field. Application of this protocol has the potential to substantially reduce the pressure on natural populations.     

Large-scale field acclimatization of Olea maderensis micropropagated plants: morphological and physiological survey

Micropropagation allows large-scale plant multiplication and germplasm preservation, representing an added value in forest breeding strategies to combat desertification and/or protect endangered species. We developed a large-scale micropropagation protocol of Olea maderensis (a native endangered wild olive of Madeira Archipelago) using OMG medium (rich in Fe, Mg and Mn) supplemented with zeatin for elongation and with NAA for rooting. We now describe the performance of micropropagated plants during five-period field acclimatization: (1) in vitro, (2) growth-cabinet, (3) greenhouse, (4) open-greenhouse, and (5) field mountain in Porto Santo Island. One hundred OG4 plants were acclimatized, showing >95% surviving rates. During acclimatization, several physiological parameters were evaluated; water content remained higher in in vitro/greenhouse conditions, decreased in field leaves. Soluble protein contents decreased during the first acclimatization periods increasing thereafter. Membrane permeability slightly increased during the field acclimatization. Chlorophylls content increased in in vitro leaves, while during acclimatization, mostly chl b decreased, increasing chl a/chl b ratio. F ₀ decreased in first acclimatization periods, increasing thereafter, while the other parameters (F _v; F _m; F _v/F _m) decreased. Nutrient contents decreased in plants transferred to poor field soil conditions, reaching values similar to mother plant leaves. Overall, with the exception of PSII fluorescence, field acclimatized plants had similar values to mother plants, showing a good adjustment to stressful field conditions. This protocol is being used in large-scale micropropagation within a reforestation program, and is an example of R&D technologies with immediate application on protection of endangered ecosystems.     

Direct Shoot Bud Formation and Tuberization from Aseptically Cultured Root Tubers of Calla Lily (Zantedeschia aethiopica L)

We describe here the development of a micropropagation protocol for mass multiplication of Zantedeschia aethiopica by using root tubers as explant. The surface sterilized root tubers produced five to six shoot-buds on semi-solid Murashige and Skoog’s (MS) medium with 10.0 mg l^?1 of 6-benzylaminopurine (BAP) and additives (50.0 mg l^?1 of ascorbic acid; 25.0 mg l^?1 each of adenine sulphate, L-arginine and citric acid). The cultures were multiplied by sub-culture of individual shoot bud produced in vitro and clumps of shoot buds generated in vitro in cultures on MS medium containing 3.0 mg l^?1 of BAP and additives. Further multiplication of propagules was achieved through tuber formation along with amplifying shoots on MS medium with 5.0 mg l^?1 of BAP. The micropropagated shoots were rooted both in vitro as well as ex vitro. Cent percent of the cloned shoots rooted in vitro within 15–18 days on hormone-free 1/2 strength MS salts with 200.0 mg l^?1 of activated charcoal. Alternatively 95–100% shoots rooted ex vitro under greenhouse conditions on soilrite after pulse-treatment with 500.0 mg l^?1 of Indole-3-butyric acid (IBA) or β-naphthoxyacetic acid (NOA) for 300 sec. The cloned plants were hardened in the greenhouse. The hardened plants were transplanted to soil for further acclimatization.     

Influence of Sugars on in vitro Rooting and Acclimatization of Carob Tree

Carob tree (Cerafoma siliqua L.) micropropagated shoots were rooted on half-strength Murashige and Skoog medium, supplemented with different types and concentrations of sugars, in order to determine the effects of sugar composition and concentration on in vitro rooting and in vivo establishment of the plantlets. Among the various sugars tested, the best rooting response was obtained with 145 mM sucrose, both in terms of rooting frequency and index of rooting. The use of filter-sterilized rather that autoclaved fructose increased root number and root length. Sugar treatment during rooting slightly influenced plantlet survival and growth during acclimatization. A reduction in the glucose concentration during rooting was beneficial for plantlet acclimatization.     

In vitro micropropagation of Basella rubra L. through proliferation of axillary shoots

In vitro micropropagation protocol for Basella rubra regeneration was tried through proliferation of axillary shoots of the potted mature plant. The improved seed germination (70%) was recorded upon 2% urea treatment. The nodal shoot segments from matured potted plant were used to initiate the multiple shoot proliferation. The shoot segments exhibited 70% shoot initiation when cultured on Murashige and Skoog (MS) medium supplemented with Indole-3-acetic acid (IAA)?+?N₆ – Benzylaminopurine (BAP) (0.25?+?2.0 mg/L) and BAP?+?Kinetin (Kin) (2.0?+?0.5 mg/L) respectively. Multiple shoots (5–6) were obtained on MS medium supplemented with BAP?+?Kin and IAA?+?BAP respectively. When compared with silver nitrate (AgNO₃) (2–40 µM) and activated charcoal (AC) (0.1–1.0%), the MS medium devoid of any plant growth regulator showed good number of shoots (5.48?±?2.42), elongation (15.64?±?2.42 cm) and root length (14.52?±?2.78 cm). Upon transferring of regenerated microshoots to MS medium, simultaneous elongation of shoots with more shoot number, shoot length and rooting was achieved during four subcultures that carried out at 6 weeks’ interval. The regenerated in vitro shoots showed 100% rooting in MS medium and also in MS medium supplemented with 0.1–1.0% AC. Hundred percent survival of micropropagated shoots well rooted was established successfully under greenhouse condition and the plants were subsequently acclimatized and transferred to the field conditions wherein 90% success rate was noted.

     

Photoautotrophic shoot and root development for triploid melon

The aim of this investigation was to establish environmental factors which promote growth and photosynthesis of melon (Cucumis melo L.) shoot buds, in vitro, and determine if photoautotrophic shoots had superior root forming ability in photoautotrophic environments. Buds from the triploid melon clone ‘(L-14×B)×L-14’ were observed for 21 days after transfer from a multiplication MS medium with 3% sucrose and 10 μM benzyladenine (BA) to a shoot development medium with 1 μM BA at three levels of sucrose in the medium (0, 1 and 3%), and light (50, 100 and 150 PPF) and CO₂ (500, 1000 and 1500 ppm) in the culture chamber. More shoot buds were observed with 3% sucrose in the medium. Increased light and CO₂ had a positive interaction with shoot proliferation. Fresh and dry weights were greatest at 3% sucrose, 150 PPF light and 1500 ppm CO₂. Shoot buds grew more slowly in sugar-free medium, but fresh and dry weight still doubled over 21 days of culture. Net photosynthetic rates (NPR) of buds were negative after four days in treatment conditions, but became positive after transfer to fresh, sugar-free medium. Two triploid genotypes of melon were (1) grown in vitro with sugar (photomixotrophic) and without sugar (photoautotrophic), (2) rooted in sugar-free media, both in a laboratory controlled environment chamber (in vitro) and a greenhouse acclimatization unit (ex vitro), and (3) compared for subsequent nursery growth in the greenhouse unit. The genotype ‘(L-14×B)×L-14’ produced more shoots than ‘(L-14×B)×Mainstream’ in both photomixotrophic or photoautotrophic conditions. ‘(L-14×B)×L-14’ rooted as well from either photoautotrophic and photomixotrophic shoots but ‘(L-14×B)×Mainstream’ rooted less frequently from photoautotrophic shoots. Seventy-six percent of the shoots in the laboratory controlled environment chamber were able to root photoautotrophically, whereas 47% of the shoots in the greenhouse acclimatization unit were rooted. Between 77% and 88% of plantlets from all treatment combinations survived transfer to the nursery. After growth in the nursery, the sizes of plants (fresh weight, dry weight, leaf area) were the same for either genotype, from either photoautotrophic or photomixotrophic shoots. Nursery plants that had been rooted in the laboratory controlled environment chamber were larger than those rooted in the acclimatization greenhouse chamber. This revised version was published online in June 2006 with corrections to the Cover Date.     

Micropropagation of Psiadia arguta through cotyledonary axillary bud culture

A micropropagation protocol for Psiadia arguta, an endangered endemic plant from Mauritius is described using 15-day old in vitro seedling explants without the radicle. MS basal medium supplemented with TDZ (0.5–1 mg/l) proved to be the most effective medium for the induction of cotyledonary axillary buds as compared to MS medium containing NAA (0.5 mg/l) or both NAA (0.5 mg/l) and TDZ (0.5–1 mg/l). In fact, after transfer to hormone free MS medium, microshoots were obtained only from seedling explants cultured on media containing only TDZ. Regenerated shoots elongated and rooted when cultured on MS8900 containing IBA (0–1 mg/l). Hormone-free MS8900 was the best medium for rooting and development of plantlets for acclimatization.     

Efficient in vitro regeneration systems for Vaccinium species

Efficient protocols for shoot regeneration from leaf explants suitable for micropropagation as well as for the development of transgenic plants were developed for blueberry (Vaccinium corymbosum) and lingonberry (Vaccinium vitis-idaea) cultivars. Nodal segments were used to initiate in vitro shoot cultures of lingonberry cultivar ‘Red Pearl’ and southern highbush blueberry cultivar ‘Ozarkblue’. In order to develop an optimized regeneration procedure, different types and concentrations of plant growth regulators were tested to induce adventitious shoot regeneration on excised leaves from micropropagated shoots of both cultivars. The effect on percentage regeneration and number of shoots per explant was investigated. Results indicated that zeatin was superior to TDZ and meta-topolin in promoting adventitious shoot formation. A concentration of 20 μM zeatin was most effective in promoting shoot regeneration in both cultivars, in case of ‘Red Pearl’ along with 1 μM NAA. Shoots were either allowed to root in vitro on medium containing IBA or NAA or ex vitro in a fog tunnel. IBA was superior to NAA for induction of root development in vitro in both Vaccinium cultivars. Ex vitro rooting under high humidity was tested with cuttings from mature field-grown plants, from acclimatized tissue culture derived plants and with unrooted in vitro proliferated shoots planted directly. It was found that in vitro shoots rooted better under fog than cuttings from the other plant sources and rooting was equivalent to that achieved in vitro.     

Influence of thidiazuron (TDZ) pretreatment of shoot tips on shoot multiplication and ex vitro acclimatization of Harpagophytum procumbens

The effects of thidiazuron (TDZ) pretreatment of shoot tips on Harpagophytum procumbens shoot proliferation and successive stages of micropropagation, i.e. rooting of regenerated shoots and acclimatization of plantlets to ex vitro conditions, were described in the present study. The best response in terms of shoot proliferation (about seven shoots/explant) and shoot length (3.2 ± 0.4 cm) was obtained when explants pretreated with 25 µmol L^?1 TDZ for 6 h were cultured on Schenk and Hildebrandt medium containing indole-3-acetic acid (IAA) (0.57 µmol L^?1) and 6-benzylaminopurine (BAP) (8 µmol L^?1). Under these conditions, a 330 % increase in shoot multiplication over TDZ non-pretreatment culture was achieved and TDZ pretreatment shoots were longer compared to those in control culture (2.6 ± 0.3 cm). The TDZ pretreatment did not affect the percentage of rooted shoots, length of roots and number of roots formed per shoot. The rooted plantlets were transplanted from in vitro to pots with soil and grown during 1 year in the greenhouse. The hardening process was difficult and time-consuming. We found that the plants developed from the TDZ pretreated culture were superior to plants from non-pretreated culture in terms of survival rate and morphological features, such as shoot length, leaf size, flowering and earlier root tuberisation. Random amplified polymorphic DNA and inter-simple sequence repeat analyses of pretreatment with TDZ plants showed genetic similarity to non-pretreatment plants. We conclude that applying the strategy of initial explant pretreatment with TDZ may be valuable for the improvement in H. procumbens in vitro propagation.