2 REVIEW OF LITERATURE
2.1 Other ornamental plants
2.2 Fruit plants
3 MATERIALS AND METHODS
3.1 Explant collection
3.2 Preparation of medium
3.3 Sterilization of explant and culture
3.5 Media used
3.5.1 Media for browning
3.5.2 Media for shooting
3.5.3 Media for rooting
3.6 Cultural conditions
3.7 Data collection
3.7.1 Shoot multiplication
22.214.171.124 Number of days to sprout
126.96.36.199 Number of laterals
188.8.131.52 Shoots length (cm)
184.108.40.206 Number of leaves/plant
3.7.2 Root initiation
220.127.116.11 Number of days to emerge roots
18.104.22.168 Number of roots
22.214.171.124 Root length (cm)
4 RESULTS AND DISCUSSION
4.1 Number of days to initiate shoots
4.2 Number of laterals
4.3 Number of leaves/explant
4.5 Shoot length
4.6 Number of days to emerge roots
4.7 Number of roots
4.8 Root length
4.10 Browning control
List of Tables
4.1 Analysis of variance for effect of Auxins and Cytokinins on shoot emergence of Rosa centifolia
4.1a Effect of Auxins and Cytokinins on shoot emergence of Rosa centifolia
4.2 Analysis of variance for effect of Auxins and Cytokinins on number of lateral shoots of Rosa centifolia
4.2a Effect of Auxins and Cytokinins on number laterals of Rosa centifolia
4.3 Analysis of variance for effect of Auxins and Cytokinins on number of leaves Rosa centifolia
4.3a Effect of Auxins and Cytokinins on number of leaves of Rosa centifolia
4.4 Analysis of variance for effect of Auxins and Cytokinins on shoot length of Rosa centifolia
4.4a Effect of Auxins and Cytokinins on shoot length of Rosa centifolia
4.5 Analysis of variance for effect of Auxins and Cytokinins on days of emergence of roots of Rosa centifolia
4.5a Effect of Auxins and Cytokinins on days of emergence of roots of Rosa centifolia
4.6 Analysis of variance for effect of Auxins and Cytokinins on number of roots of Rosa centifolia
4.6a Effect of Auxins and Cytokinins on number of roots of Rosa centifolia
4.7 Analysis of variance for effect of Auxins and Cytokinins on root length of Rosa centifolia
4.7a Effect of Auxins and Cytokinins on root length of Rosa centifolia
List of Figures
4.1 Shoot induction from nodal segment of Rosa centifolia
4.2 Shoot proliferation from nodal segments of Rosa centifolia
4.3 Root induction from the explant of Rosa centifolia
4.4 Acclimatization of Rosa centifolia
4.5 Acclimatization of Rosa centifolia
4.6 Effect of different treatments on browning of MS media
The present study was conducted for regeneration of Rosa centifolia and reduction of oxidative browning. Shoot tips with axillary buds were used as explant. The explants were surface sterilized with 70% alcohol for 4 minutes and 5% bleach for 4 minutes followed by three washing with autoclaved double distilled water After that the explants were cultured on MS media (Murashige and skoog,1962) supplemented with different concentration of BAP (Benzyladenine) and NAA (Naphthalene acetic acid). Effect of different concentrations of BAP (1.5 mg/l, 3 mg/l) alone and in combination with NAA (0.5 mg/l) for shoot induction. BAP and NAA (3.0 mg/l +1.5 mg/l respectively) gives the good results for shooting. the effect of NAA (1.5 mg/l, 3 mg/l) alone and in combination with BAP (0.5 mg/l) for root induction was examined. NAA and BAP (3.0 mg/l +1.5 mg/l respectively) gives the good results for rooting. After rooting plants were acclimatized and successfully transferred to the greenhouse. To control the oxidative browning in Rosa centifolia different treatments (charcoal, charcoal + running water, stirring in antioxidant solution of citric acid and ascorbic acid) were used. But the charcoal with the running water gives the good results.
CHAPTER 1 INTRODUCTION
Rose belongs to family Rosaceae and subfamily Rosoideae, is only found naturally occurring in the temperate parts of the northern hemisphere and the distribution of species is mainly divided into four regions: Europe, America, Asia and the Middle East, with the greatest diversity of species found in western China (Phillips and Rix 1988). It is believed that roses grew in the wild from prehistoric times and fossils thought to be over thirty-million years old, found in Colorado and Oregon, in the USA. The genus Rosa contains more than 1400 cultivars and 150 species (Gault and Syngy, 1971). Roses are one of the most important flower crop in the world (Short and Robert, 1991). The rose is the quintessential romantic plant. Since ancient times, they have been associated with love, courtship, beauty and romance. The flavours and fragrance of rose is unmatched and most sought after in the food and cosmetic industry. Rose oil, rose water, rose concrete and rose absolute are some of the most important products that are in great demand (Kaur et al., 2007). Roses are used for decorative purposes. Roses add beauty, charm and fragrance in nature. They also used on different religious and traditional occasions, as cut-flower and a good source of rose oil and vitamins C (Marchant et al., 1996).
Rosa centifolia is an important landscape plant used in parks, gardens and houses. Rosa centifolia is very important among oil producing species of roses (Tucker and Maciarello, 1988) The rose oil and its petals have commercial value and used in perfume industry, food items and medicines.
Rosa centifolia is commercially propagated by asexual methods that are sucker, hard wood cutting, semi hard wood cutting, budding and grafting (Horn, 1992). Although propagation by vegetative means is a predominant technique in roses, yet it does not ensure healthy and disease-free plants. Moreover, dependence on season and slow multiplication rate is one of the other major limiting factors as limitation of stock plant and prolonged production time in conventional propagation. It has also been observed that plants raised from these methods are infected with different diseases that affect flower production and quality, and ultimately their market value is decreased (Norton & Boe, 1982). The in vitro culture of roses can be performed by using axillary buds as explants ( Khosh-Khui and Sink, 1982; Arnold et al., 1995; Marcelis Van Acker and Scholten, 1995; Gudin, 2001)
Plant tissue culture is the science of growing plant cells, tissues or organs isolated from the mother plant, on artificial media in aseptic conditions. Propagation of plants through tissue culture has become an important and popular technique to produce crops that are other wise difficult to propagate conventionally by seed and vegetative means.
The history of rose tissue culture dates back to 1945, when Nobecourt and Kofler succeeded in obtaining callus and roots on the explanted buds. In the year 1946, Lamments for the first time reported the use of embryo culture in rose breeding. Studies were initiated by Nickell and Tulecke (1959) and Weinstein et al. (1962) to culture cells, cell suspension and calli with a view to understand differentiation and regeneration. The earliest references of rose micropropagation were those of Jacob et al. (1969, 1970a,b) and Elliott (1970) in R. hybrida cv. Superstar and R. multiflora, respectively.
In tissue culture specialized and mature cells are manipulated to give rise the multiple copies of the parent plant under optimum aseptic environmental conditions. It offers many unique advantages over conventional propagation methods such as rapid multiplication of valuable genotypes, expeditious release of improve varieties, production of disease free plants, healthy, non seasonal production (a round the year), (Dhawan and Bhojwani 1986) germplasm conservation and facilitating their easy international exchange. Tissue culture technique used for rapid and mass propagation of several plant species particularly ornamentals. Commercialization of micropropagation has found its best application in ornamentals (Chaturvedi et al., 1994). In vitro propagation offers rapid propagation of plants in limited space and at any time due to control conditions. There are several advantages of plants produced through micropropagation over conventional methods. Plants produced through micropropagation technique are disease free, true to type and can be produced round the year. Micropropagated plants are well suited for cut flower production as they are more compact (Onesto et al., 1985), branch better and sometimes yield more flowers (Reist, 1985).
All woody plants including roses have the problem of browning, when propagated through in vitro techniques. Browning is due to release of phenolic compounds from the cut surfaces of the explants (Bhat & Chandel, 1991). Browning of excised plant tissues as well as nutrient media occurs frequently and remains a major basis for recalcitrance in vitro. Browning can result into cessation of growth or development and ultimately death of the explants. The severity of browning has varied according to species, tissue or organ, developmental phase of plant, age of tissue or organ, and nutrient medium and other tissue culture variables. The discoloration is associated with precocious death or noticeably retarded development. Browning is one of the important factors which limits the growth of roses in vitro culture. Browning can be controlled by adding substances like activated charcoal (Skirivin and Chu, 1979). Activated charcoal also increase the absorption of compounds such as the phenolics and their oxidates, auxins [indole- 3-acetic acid (IAA), naphthaleneacetic acid (NAA), indoel-3-butyric acid (IBA)], cytokinins [benzyladenine (BAP)], and hormones (having heterocyclic and unsaturated ring structures (e.g. kinetin), the main problem for slow and less growth of Rosa centifolia in tissue culture is oxidative browning. To control oxidative browning different treatments (charcoal, charcoal and running water, citric acid and ascorbic acid) can be used.
The status of plant tissue culture in Pakistan is miserably poor as compared to the other developed countries. For the establishment of an effective plant propagation system should focus on explant type, nutrient medium composition and environmental growth conditions.
The objective of this study is to micropropagate the Rosa centifoia and to overcome problems that inhibit its production. Thus disease free plants in mass quantity will be produced.
CHAPTER 2 REVIEW OF LITERATURE
In vitro propagation has revolutionized commercial nursery business. Significant features of in vitro propagation procedure are its enormous multiplicative capacity in a relatively short span of time; production of healthy and disease free plants; and its ability to generate propagules around the year.
Skirvin and Chu (1979) cultured shoot tips of the glass house rose cv. ‘forever yours’ about 2 cm long on Murashige and Skoog (MS) medium with vitamins, 2 mgL- 6-benzylamino purine, 0.1 mgL- NAA and 100 mgL- myoinositol. About 25% shoot tip developed multiple shoots from axillary buds within 5 weeks. Other shoot tips were placed on MS medium lacking BA but containing vitamins, 100 mgL- myoinositol and NAA at various rates. Within three weeks 11-20% shoots initiated roots on 1-3 mgL- NAA. Young rooted plants were successfully transferred directly from culture tubes to clay pots containing vermiculite and within 3-5 weeks they were large enough to transfer to soil. About 50% of all the plantlets survived to maturity.
Hasegawa (1980) concluded that in vitro derived shoot tips of ‘Improved Blaze’ rose (Rosa hybrida L.) proliferated on a medium containing MS salts, thiamine HCL (0.5mgL-), pyridoxine HCL (0.5mgL-),sucrose (30gmL-), glycine (2.0mgL-) Bacto agar(8mgL-), IAA (0.03mgL-) and 6- benzylaminopurine (1.0, 3.0, 10 mgL-). A 6-fold multiplication was obtained after 10-14 days in the basal medium with out growth regulators. Roots were initiated and were successfully transferred to soil.
Davies (1980) studied in vitro propagation of roses by using Murashige and Skoog medium supplemented with 0.4 mgL- NAA, 2 mgL- BA, 4% sucrose and 0.1 mgL- GA3. Rate of multiplication was between 3 and 5 after 4 weeks of period attained over a series of subcultures. Rooting was achieved under standard greenhouse conditions.
Marting et al. (1981) propagated greenhouse roses, out door floribundas, miniatures and a range of rootstocks on a modified Murashige and Skoog (1962) medium containing different levels of auxins, cytokinins and gibberellins. Over 2000 micropropagated plants on their own roots were compared for three years with traditionally grafted control plants. Micropropagated plants produce 10% more flowers than the control in the first year and 20% more in the second and third year.
Avramis et al. (1982) reported that Rosa indica major wildly used in France as a rootstock for greenhouse cut rose production is difficult to propagate because of its susceptibility to low temperature damage. In vitro culture of node or shoot tip explant produced shoots on Murashige and Skoog (1962) medium with 2 mgL- BA and roots on MS medium with 0.1 mgL- NAA.
Khosh-Khui and Sink (1982) studied the effect of combination of auxin sources and combinations, relative to root formation of (Rosa hybrida L.) Bridal Pink propagated in vitro. Indol acetic acid (IAA) and 6- benzylaminopurine (BA) alone were almost ineffective to stimulate rooting. While concentrations of NAA and IBA, NAA and IAA were equally affective in stimulating rooting but better root quality in NAA and IBA combination was obtained.
Khosh-Khui and Sink (1982) studied the micropropagation comparisons between two Rose hybrida cvs. ‘Tropicana’ and ‘Bridal Pink’ and two old world species. Species variation were observered for growth regulators requirement and rate of multiplication, not only between the (Rosa hybrida L.) cvs. But also between the old world species. It was concluded that 2 mgL- BA was optimum for hybrid roses + 0.05 and 0.10 mgL- NAA for cvs. tropicana and bridal pink respectively. Both rooting ability and acclimatization to the planting medium were lower in old world species than in the (Rosa hybrida L.) cvs.
Barve et al. (1984) cultured axillary vegetative buds and shoot tips of cultivar ‘Crimson-Glory’ and ‘Glenfidich’ on modified White or Murashige and Skoog medium. Multiple shoot formation occur on MS medium containing kinetin (0.2 mgL-) and BA (0.5 mgL-) good rooting (60%) was obtained on the proliferated shoots by lowering the MS concentration to half strength and by adding IAA + IBA + indole propionic acid, each at 0.5 mgL-. Then plantlets were then transferred to pots in greenhouse and later to field. In the field, the plants showed a high degree of uniformity in growth, petal number, flower size and number.
Cai et al. (1984) cultured stem segments each with an axillary bud, on MS medium plus BA at 1-3 mgL- and NAA 0-0.1 mgL-. After 2 weeks, the axillary buds started to grow and after 5 weeks of transplantation on MS medium supplemented BA (1-3 mgL-), a rosette of shoot formed. Proliferation and growth of shoot was different with different cultivars. Most of the shoot cultured on half MS strength medium + NAA (0.5-1 mgL-) for root induction, within 10-15 days produced roots. The rooted plantlets transplanted to pots and survival rate was 90% and flowered within two months.
Curir et al. (1985) micropropagated rose cultivars Bellona, Bingo, Candia, Cocktail and Sonia on modified Murashige and Skoog (1962) medium. Three days of culture in the presence of activated charcoal, followed by transfer to fresh basal medium, promoted the growth of shoots. Thiamine (2 mgL-) and myoinositol (100 mgL-) promoted shoot proliferation and NAA (0.8 mgL-) with low sucrose (15 mgL-) promoted rooting.
Proft et al. (1985) cultured rose cv. ‘Blue Sette’ plantlets on Murashige and Skoog (1962) nutrient medium. When NAA (1.0 mgL-) was added to the basic medium to stimulate shoot growth. Adding 2 mgL- IAA in combination with 2 mgL- BA markedly stimulated lateral shoot growth.
Reist (1985) compared cut flower yield from own rooted rose bushes established at the same time from cuttings or in vitro propagated plants. Flowers were two weeks earlier in plants grown from cuttings; in vitro grown plants gave 20 percent higher yield from plants grown in the field.
Collet and Le (1987) tested micro cuttings of rose cv. Cosette taken from proliferated shoot in vitro for their ability to produce adventitious roots with respect to two different modes of initiation :(1) long initiation pretreatment (7 days culture on mineral salt medium + sucrose + IBA or IAA) OR(2) brief induction treatment (16 hour tip in 10- M IAA solution). The best result (rate of rooting, number of roots and absence of callus) were achieved through the 'brief induction pretreatment’ with IAA supplemented at the cut end of cutting.
Kodytek (1887) cultured shoot explants of the rose cv. Sonia on modified Murashige and Skoog medium containing growth regulators. Shoot proliferated readily in the presence of 1.0 mg BA + 0.02 mgL- IAA but after 6 weeks aging become apparent and the rate of new shoot production decreased. Rooting percentage was highest on a medium containing 1.0 mgL- IAA and was higher in shoots grown with IAA alone than in shoots grown with IAA + BA..
Mederos and Enriquez (1987) determine the factors effecting the shoot tips and axillary buds of ‘Golden Time’ roses in vitro. MS medium supplemented with IAA, NAA and/or IBA used alone or in combination with BA on root formation. Shoot proliferation in the presence of BA and the root formation was best in medium containing BA or BA + IAA.
Valles and Boxus (1987) studied the micropropagation of 11 Rosa hybrida cultivars. Quoirin-Lepoiure salt solution was used and different growth regulates were tested. IBA at 0.1 mgL- reduce proliferation where BA at 1.0 mgL- enhances it. GA3 at 1.0 mg L- enhanced axillary branching. Preliminary results on rooting showed different hormonal requirements of some cultivars.
Mederos and Enriques (1988) determine the factors affecting shoot tips and axillary bud growth. MS medium supplemented with IAA, NAA and IBA used alone or in combination with BA on root formation. The findings are presenting and the data tabulated. Shoot proliferation was best in the presence of BA and the root formation was best in medium containing BA or BA + IAA.
Douglas et al., (1989) initiated cultures from shoot tips and nodal segment explants of 4 rose cultivars. Average shoot proliferation rates were 3.1, 1.3 and 2.5 shoots per culture respectively, on MS medium supplemented BA (1.0mgL-), NAA (0.1mgL-) and GA3 (0.1mgL-). The proliferation rate of cv. Queen Elizabeth was significantly increased by using long shoots (>2cm in length) and by subculturing shoots on fresh medium every 3 weeks.
Rout and Debata (1989) cultured axillary buds on MS medium at 25±2C. Shoot multiplication experiments were carried out on MS medium supplemented with BA and GA3. The highest number of shoots per explant (3.5±µ0.74) was obtained with 0.1 mgL- BA. BA + GA3 produced more robust shoots compared with BA alone. Explant survival was 80-90% for all treatments.
Campose and Pais (1990) cultured shoot tips and axillary buds of three pot rose cv. Rosamini on MS medium containing 30gL- sucrose, 8gL- agar and supplemented with 1 mgL- BA and 1-4 mgL- IBA. Multiplication rates of 6-7 fold were reached when shoots were subcultured every 4 weeks, and these rates decreased with prolonged maintenance of shoots without subculturing. Root induction was achieved with half strength MS medium containing 20gL- sucrose, 6gL- agar and supplemented with 1 mgL- IBA. Root elongation occurred on the similar medium but without IBA.
Dohare (1991) developed a method for the propagation of Rosa hybrida cv . Super Star. Axillary buds, collected from the middle of the actively growing branches, proliferate multiple shoots on MS medium supplemented with 2 mgL- BAP, 0.1 mgL- NAA and 0.5 mgL- GA3. A 5- fold multiplication was obtained after 6 weeks of culture. Roots were obtained from 85% of these shoots within 7-10 days after transfer to MS medium supplemented with 0.5 mgL - IBA.
Suvorova et al., (1991) reported that in vitro propagation was carried out with groups of ornamental roses comprising 19 varieties and 7 seedlings, including 5 varieties of the floribunda type and 14 of the ‘Hybrid Tea’ type. There were difference between varieties in the optimum medium for successful culture and root development.
Vajaya and Satyanarayana (1991) stated that lateral bud of rose (cv. Andhra Red) were cultured on three types of media, of which MS proved the most suitable for lateral shoot proliferation. BA was most effective growth regulator in stimulating shoot proliferation. An increase in the shoot length was obtained by adding IAA to the medium as compared to BA in MS medium alone. The significant difference in the number of shoots produced per culture in relation to different auxin treatments.
Bhat (1992) described a procedure for micropropagation of rose cv. ‘Super star’ from lateral bud explant. Explants were established on the culture medium supplemented with different levels of BA, nephthoxyacetic acid (NAA) or IAA. The highest rate of culture establishment (99.4%) and growth were obtained with 2mgL-and 0.1mgL- NAA. Shoot were obtained using a shoot multiplication medium supplemented with 0.1mgL- NAA and 0.5mgL- GA3.
Li (1992) reported that inclusion of activated charcoal in the medium encouraged but formation and root developed in the presence of 2 mgL- ABT (1, aminobenzotriazole), 87.7% of the shoots form roots i.e. 28.7% and 26.0% more than in the presence of NAA and IBA, respectively. Under the dry conditions of the north western China, 18.9-96.7% of the plantlets survived to soil beds.
Rahman et al. (1992) reported that basal medium strength, auxin, sucrose and agar concentration, PH, photoperiod and culture room temperature substantially affected root development in vitro by proliferating rose cv. Tajmahal shoots. Half strength MS medium fortified with 0.1 mgL- NAA and 0.5 mgL- IAA and containing sucrose and 6 gL- agar, 16 hour light/day and a temperature of 28C have better responses for optimum in vitro rooting of the excised shoots as compared with full strength MS medium.
Choudhary (1993) developed a method for a rapid propagation of rose cv. Priyadarshini from axillary bud explants and found that maximum shoots (5.7-5.8 per explant after 80 days) were proliferated on MS medium supplemented BA (2.5- 5 mg/L-). Kinetin did not stimulate shoot proliferation. Rooting of shoot was most successful on half strength MS medium supplemented with NAA at 0.5 mgL-.
Chu et al, (1993) Studied the growth of miniature rose (Rosa chinensis Jacq. ‘Minima’) shoots cultured on liquid medium was greater relative to those cultured on two-phase (solid + liquid) medium or solid medium alone. Shoot multiplication ratio (number of multiple shoots per explant per subculture) on liquid medium was higher with 17.8–26.6µM 6-benzyladenine at compared to that at 0–8.9 µM. Shoots grown on 30 ml or more of liquid medium had a higher multiplication ratio than those grown on 10 or 20 ml. The growth and multiplication ratio increased when the culture period was extended from 3 to 6 weeks, although plantlets began to exhibit some chlorosis after the 6th week. These conditions were maintained over four subcultures for cultivars Abbildung in dieser Leseprobe nicht enthaltenBaby KatieAbbildung in dieser Leseprobe nicht enthalten, Abbildung in dieser Leseprobe nicht enthaltenLavender JewelAbbildung in dieser Leseprobe nicht enthalten, Abbildung in dieser Leseprobe nicht enthaltenRed SunblazeAbbildung in dieser Leseprobe nicht enthalten and Abbildung in dieser Leseprobe nicht enthaltenRoyal SunblazeAbbildung in dieser Leseprobe nicht enthalten, with no significant change in multiplication ratio over time
Savamal and Singh (1994) reported that the shoot tips and nodal segments of rose cv. Sonia proliferated on MS medium supplemented with various growth regulators. Nodal segments proliferated higher number of shoots per explant than shoot tips. The optimal combination of growth regulators for shoot proliferation was 2.0 mgL- BAP + 0.1 mgL-IAA + 0.1mgL- GA3. Shootlets when transferred individually into half strength MS basal medium supplemented with 0.2mgL - NAA + 0.2 or 0.5 mgL- IBA produced roots.
Barna and Wakhlu, (1995) studied the micropropagation of Rose (Rose hybrida L.) plant by axillary shoot proliferation method. Maximum number of microshoots per shoot tip explant were obtained on MS medium supplemented with 5 to 10M thidiazuron (TDZ). The microshoots formed rooted plants on MS hormone-free medium. No difference in the rooting of microshoots produced on medium containing TDZ or N6-benzyladenine was observed. The regenerated plants were successfully transplanted to the field and appeared similar to the parent plant in morphologic features.
Marcelis-van Acker and Scholten, (1995) developed an in vitro model system to grow axillary buds into shoots, which are morphologically comparable to those grown in vivo.. A low concentration of benzyladenine (BA), sugar (preferably glucose), and a cultivar-dependent concentration of a suitable agar was necessary to obtain a complete shoot. The size of the in vitro shoot positively correlated with the size of the explant. The presence of the petiole inhibited the outgrowth of lateral buds, which were already present in the inoculated bud. Using this in vitro system the growth potential of axillary buds, apart from influences of other plant parts, can be studied.
Podwyszynska and Olszewski, (1995) developed a protocol for shoot cultures of Rosa hybrida cultivar ‘White Gem’, Cordyline fruticosa cultivar ‘Atoom’ and Homalomena cultivar ‘Emerald Gem’ were cultured in vitro on proliferating modified Murashige and Skoog (MS) media solidified with different gelling agents: 7 g l− Bacto-Agar (Difco), 7 g l− Agar Purified, 3.5 g l− Agargel, 2 g l− Phytagel and 2 g l− Gelrite. Rose shoots were also cultured on media containing a double concentration of Ca and Mg. The gelling agent did not influence the multiplication rate of any species significantly. However, gellan gum increased the shoot length of homalomena and rose and the fresh weight of cordyline shoots. The use of higher levels of Ca and Mg in the Bacto-Agar medium improved the quality of rose shoots, and in Gelrite medium increased the rose multiplication rate. Chemical analysis of the plant material, derived from the Bacto-Agar MS media, showed that the differences in N, P and K contents between species did not exceed 15%.
Ghashghaie et al., (1996) studied micropropagation of Rose (Rose hybrida cv. Madame G. Delbard) on agar at varying concentrations: 0, 4, 5.5, 6, 7.5, 8, 9 and 15 g l−. Water potential of the medium was lincar function of agar concentration, and gel strength increased linearly from zero as agar concentration increased from 5 to 15 g l−. Except for liquid medium, where shoot proliferation was imparied by vitrification, fresh and dry weights and number of total shoots decreased linearly with increasing agar concentration. At low agar concentration, where water potential of the gelled medium was high. Number of usable (elongated) shoots was significantly higher at a moderate level of agar, namely 7.5 g l−. This can be explained in terms of the antagonistic actions of cytokinin and water on shoot elongation. At low agar concentration, leaf water potential and absolute water content were high. At high agar concentration, under gel water deficit, leaf water potential and absolute water content were low but an osmotic adjustment maintained turgor pressure.
Habib et al. (1996) cultured rose explants in vitro for multiple shoot developed and subsequent rooting. Multiplication of shoot was carried out from shoot apices and nodal segments on MS medium supplemented with 2.0 mgl- BAP, + 0.1 mgL- BAP + 1.0 mgL- Kinetin and 2.0 mgL- BAP + 0.5 mgL- 2, 4-D. The highest number of multiple shoots appeared in presence of BAP at 2.0mgl- within 8 to 10 weeks.
Ma et al, (1996) Studied that several rose species (Rosa rugosa, R. wichuraiana, R. setigera, R. laevigata, R. banksiae, R. roxburghii, R. odorata) and interspecific hybrids were cultured to determine the appropriate concentrations of nutrients and growth regulators for shoot proliferation and root initiation. Cultured shoot tips and lateral buds from different genotypes proliferated multiple shoots on a basal MS medium supplemented with 0 μM to 17.8 μM 6-benzyladenine (BA) and 0 μM to 0.54 μM naphthalene, acetic acid (NAA). The ability of the explants to proliferate shoots and initiate roots was affected by the genotype, the nodal position of the explant, the strength of the MS basal salts, and the growth regulators used. Most species had the highest shoot proliferation when cultured on basal MS medium supplemented with 8.9 μM BA, but the degree varied by species. Root development was enhanced by lowering the concentration of MS salts. Rooting was improved by supplementing the media with 11.4 μM indole-3-acetic acid (IAA) or by giving them a 7-d dark treatment at 10°C.
Ara et al. (1997) cultured 1st and 2nd nodes of young and old shoot tips for multiple shoot regeneration on MS medium supplemented with 1.0 mgL- BAP. Multiple shoot were subcultured on the same medium and then transferred to half strength MS medium containing different concentrations and combinations of IBA, IAA and NAA for root deveopment. IBA (0.5 mgL-) was found to be suitable for root induction.
Dobariya et al. (1997) concluded that cultures of rose cvs. Super Star, Dilkhush and Glandiator were successfully established non Murashige and Skoog (MS) basal medium using nodal explants with an axillary bud. Muliple shoot formation was evaluated on MS medium with different growth regulator concentrations. They produced highest percentage of cultures developing multiple shoots and a good multiplication rate. Micropropagated shoots were rooted on half strength MS medium (60-70% rooting).
Salehi et al., (1997) studied the explants of a miniature rose (Rosa chinensis Jacq. var. minima Rehd hybrids) cultivar ‘Baby Masquerade’to find the most suitable procedure for their disinfection. Single-node explants were dipped into a sterilized solution of different antibiotics (gentamicin, ampicillin, tetracycline or amoxicillin) with different concentrations and duration before and after surface sterilization. In general, using antibiotic solutions before surface sterilization was unsuccessful. Dipping in a 100 mgL-l solution of gentamicin or ampicillin after surface sterilization resulted in the highest percentage of disinfected explants (90% and 84%, respectively). This simple procedure may be a more convenient and nontoxic method than including antibiotics or other germicides in the medium.
Ganga (1998) studied the effect of MS and woody plant (WP) media supplemented with growth regulators BA, GA3 and NAA, individually and in combination, on multiple shoot induction from axillary bud of rose cultivar Andhra Red. The highest percentage of bud break (83%), the earliest bud break (6.1 days) and the highest number of microshoots (4.83) were recorded in MS medium supplemented with 2.0 mgL- BA, 0.1 mgL- GA3 and 0.1 mgL- NAA in combination and alone.
Jayasree et al. (1998) reported that early differentiation of axillary buds of Rosa hybrida was the best in MS medium supplemented with 2 mg L- or 4 mgL- Kinetin or 2mgL- BAP, individually or in combination with 0.1mgL- NAA. Induction of multiple shoots was the highest with 4mgL- BAP. Only single shoots were developed on cultures supplemented with kinetin. The longest shoots with the highest number of leaves developed on medium supplemented with 2 mg- Kinetin followed by 2mgL- BAP.
Rohman et al. (1998) described the methods for in vitro propagation of roses cv, White Bengal Rose. The best conditions for shoot proliferation were half- strength MS medium supplemented with BA and 2mgL- Kinetin. The best rooting of shoots was observed in the presence of 2mgL- IBA.
Wilson and Nayar (1998) described that surface sterilization, stage of explant, media for culture establishment and multiple shoots induction were standardized for in vitro propagation of rose cv. Folklore. Treatment with 0.08% mercuric chloride for 12 min. was optimum for the surface sterilization of axillary bud explants. Axillary buds of 1.0 cm length exhibited the best response. MS medium supplemented with BAP at 2.5 mg L-, 2, 4-D at 0.5 mgL- was suitable for culture establishment.
Li et al. (1999) reported that, shoot proliferation of climbing rose cultivars was the best in MS medium supplemented 0.5-1 mgL- BA and 0.01 mgL- NAA. 100% rooting was achieved on half- strength medium supplemented with 0.2mgL- IBA.
Marcelis-van Acker et al. (1999) developed an in vitro system to grow axillary buds into shoots, which are morphologically comparable to those grown in vivo. In the development of the shoot three stages can be distinguished: sprouting of the bud, unfolding of leaves already present in the bud, and new formation of leaves and a flower bud. They used low concentration of benzyladenine (BA), sugar, and a cultivar dependent concentration of a suitable agar was necessary to obtain a complete shoot. The size of the in vitro shoot positively correlated with the size of the explant. The presence of the petiole inhibited the outgrowth of lateral buds, which were already present in the inoculated bud. Using this in vitro system the growth potential of axillary buds, apart from influences of other plant parts, can be studied
Nakano et al. (1999) observed adventitious shoots from both leaf and petiole segments of the `rose-formed' strain of hybrid tuberous begonia on MS media containing 0.54 µM NAA and 0.44 µM BA. Shoots were efficiently multiplied by shaking shoot-forming leaf segments in a liquid medium containing 0.54 µM NAA and 0.44 µM BA, while shoot growth was stimulated by shaking them in a liquid medium without plant growth regulators. Plantlets were obtained by rooting the elongated shoots on half-strength MS media containing 0.54 µM NAA and solidified with 8 g l- agar or 2 g l- gellan gum, and successfully transferred to the greenhouse. Regenerated plants grew into the flowering stage and showed no apparent morphological alterations.
Chakrabarty et al. (2000) reported that nodal explants 1.5-3cm long of 10 rose cultivars were grown on MS medium containing various levels of IAA and BAP. Multiple shoots were produced on medium containing 2.0mgL- BAP and 0.2mgL- IAA. The in vitro grown propagules were rooted in quarter strength MS medium supplemented with 0.1 mgL- IAA + 0.1mgL- IBA. Rooting was evident in 8 cultivars whereas two cultivars (Windy City and Doris Tysterman) did not respond.
Kapchina-Toteva et al, (2000) indicated the effect of purine (BA) and phenylurea (CPPU) cytokinins on apical dominance release in vitro cultured Rosa hybrida L., cv. Madelon and Motrea was evaluated. Cv. Madelon shows stronger natural apical growth and fewer branches than cv. Motrea in vivo and in vitro. The effects under three conditions, without the addition of the auxin IBA, in the presence of IBA, and in material pretreated with a pulse of IBA. Results were scored weekly for 4 weeks. BA and CPPU stimulated axillary bud break, and higher numbers of open buds were recorded in the presence of CPPU. When CPPU cytokinin was added to culture medium, physiologic features such as bud sprouting and shoot fresh and dry weight were enhanced. CPPU was also highly efficient for overcoming IBA inhibition of bud outgrowth. Different cultivar responses were observed.
Mohan et al. (2000) developed rapid and efficient micropropagation protocols of essential oil-producing species of rose, Rosa damascena and Rosa chinensis. They tested 12 media combinations, in which basal medium (MS) was supplemented with different concentrations of BAP and IAA, used to induce adventitious shoots regeneration from nodal explants of Rosa damascena and Rosa chinensis.