Environmental control for the large-scale production of plants through in vitro techniques

Leafy or chlorophyllous explants of a number of plant species currently micropropagated have been found to have high photosynthetic ability. Their growth and development have been promoted on sugar-free medium rather than on sugar-containing medium, provided that the environmental factors, such as CO₂ concentration, light intensity and relative humidity, are controlled for promoting photosynthesis and transpiration of explants/shoots/plantlets in vitro. Thus, environmental control is essential for promoting photosynthetic growth and development of in vitro plantlets. Several types of sugar-free (photoautotrophic) culture systems for large-scale micropropagation of plants have been developed. Advantages of sugar-free over conventional (heterotrophic or photomixotrophic) micropropagation systems are as follows: growth and development of plantlets in vitro are faster and more uniform, plantlets in vitro have less physiological and morphological disorders, biological contamination in vitro is less, plantlets have a higher percentage of survival during acclimatization ex vitro, and larger culture vessels could be used because of less biological contamination. Hence, production costs could be reduced and plant quality could be improved significantly with photoautotrophic micropropagation. Methods for the measurement and control of in vitro environments and the beneficial effects of environmental control on photosynthetic growth, development, and morphogenesis in large-scale production of micropropagated plantlets are presented. This revised version was published online in June 2006 with corrections to the Cover Date.     

Photosynthesis of plant régénérants. Specificity ofin vitro conditions and plantlet response

Regenerants from tobacco(Nicotiana tabacum L. cv. White Burley) leaf segments cultivatedin vitro in vessels with solid agar medium under usual conditions (plantlets) grew under very low irradiance (I = 40 μxmol m?2 s?1), very high relative humidity (more than 90%) and decreased CO₂ concentration (c_a) during light period. In comparison with seedlings of a similar number of leaves and similar total leaf area grown in sand and nutrient solution, the plantlets had lower dry mass of shoots and roots per plant and thinner leaves almost without trichomes and epicuticular waxes. Due to a low transpiration rate under high relative humidity the water potential of plantlet leaves was higher than that of seedling leaves and the difference in water potential between leaves and medium was lowei. The rate of water loss from leaves detached from plantlets was considerably faster than that from seedlings under the same conditions (I = 110 μrnol m^?2; s^?1, temperature 30 °C, relative humidity 50 %). Net photosynthetie rates (P_n) of leaves of plantlets and seedlings measured under saturating I, natural ca and the leaf temperature 20 °C were similar, nevertheless the shape of curves relating Pn to c» indicated some differences in photosynthetie parameters(e.g. saturation of Pn under lower ca> higher CO2 compensation concentration in plantlets than in seedlings). Similarly compensation and saturating I were lower in plantlets than in seedlings. The shape of transpiration curves as well as the expressive linear phases of P_N(ca) and P_N(I) curves of plantlet leaves indicated ineffective stomatal control of gas exchance. These results were confirmed by microscopic observations of stomatal movementsin situ     

Quality of micropropagated plants—Vitrification

Summary Vitrification-Hyperhydrous shoot development, effects the survival and quality of several micropropagated plants ex-vitro. The leaves which are the immediate organ to be affected, exhibit abnormal morphology and physiology. Leaf malfunction is apparently a stress response to very rich media and high relative humidity. The understanding of the underlying mechanism of vitrification and its control in vitro can contribute to a more efficient micropropagation. Vitrification was found to be associated with elevated ethylene production which was related to hypolignification and poor cell wall development. Liquid and low agar media induced callose formation along with reduced and disoriented cellulose biosynthesis, manifested also in non-functioning guard cells. Malfunctioning stomata, in addition to defective cuticle contributed to increased transpiration and desiccation of in vitro formed leaves. The activity of various enzymes, associated with cell wall synthesis, was low and total proteins in normal leaves was higher than in vitreous ones. Various measures were found to reduce vitrification; lowered matrix and water potential in the medium, reduction in RH, low NH ₄ ⁺ , changes in Ca⁺⁺ levels and the removal of ethylene. These measures improved leaf morphogenesis, survival and the quality of several micropropagated plant species. Presented in the Session-in-Depth Transition of Plants From Culture to Establishment In Vivo,“ at the 41st Annual Meeting of the Tissue Culture Association, Houston, Texas, June 10–13, 1990.     

Comparison of somatic embryogenesis-derived coffee (Coffea arabica L.) plantlets regenerated in vitro or ex vitro: morphological,mineral and water characteristics

Coffea arabica L. plantlets obtained ex vitro after sowing somatic embryos produced in a bioreactor in horticultural substrate were compared with those obtained in vitro from the same embryo population under conventional culturing conditions on semi-solid media. The intensity and quality of aerial and root system development were compared. Shoot emergence was more efficient in vitro but rooting frequencies were low. In contrast, all ex vitro-regenerated embryos rooted. The cotyledon area of mature embryos produced in a bioreactor positively affected plantlet development when regeneration was carried out ex vitro. Embryos with an intermediate cotyledon area (0.86 cm2) had the highest rates of plant conversion ex vitro (63%), and also resulted in vigorous plantlets. Mortality was higher in nursery conditions, but better plant development was obtained. The quality of plantlets produced under ex vitro conditions was reflected in better growth of the aerial and root systems, and also by similar morphological, mineral and water status characteristics to seedlings. Unlike roots formed on semi-solid media, those produced in soil were branched, fine (30-50% had a diameter of less than 0-5 mm) and they bore root hairs. Leaves of plantlets regenerated ex vitro had a histological structure similar to that of seedling leaves, and a lower stomatal density (100 vs. 233 mm-2). Moreover, they were more turgid, as indicated by higher pressure potential (psiP) (0.91 s. 0.30 MPa) and relative water content values (97 vs. 93%). Furthermore, under in vitro conditions, leaves had larger stomata which were abnormally round and raised. Direct sowing of germinated somatic embryos resulted in the rapid production of vigorous plantlets under ex vitro conditions, whilst removing the need for problematical and costly conventional acclimatization procedures.     

Physiological changes and growth of micropropagated chile ancho pepper plantlets during acclimatization and post-acclimatization

Little is known about physiological changes that occur with micropropagated chile ancho pepper (Capsicum annuum L. cv. San Luis) plantlets during acclimatization. Plantlets were transferred to ex vitro conditions to study selected physiological changes and growth performance during acclimatization and post-acclimatization. The physiology of the plantlets was characterized by measuring leaf gas exchange and water status. Plant growth was determined by assessing plant height, leaf number, total leaf area, relative growth rate (RGR), and leaf, root, and stem dry matter (DM). Chile pepper plantlets became acclimatized within 6 days after transplantation. During this period, physiological adjustments occurred, which were critical for plantlet survival. After initial ex vitro transplanting, plantlets experienced water deficit [leaf wilting and reduced relative water content (RWC)], which corresponded with reduced stomatal conductance (g _s) and transpiration (E), and an increase in stomatal resistance (r _s). Thus, leaf stomata that developed in vitro were functional ex vitro. Because of this stomatal control, plantlets minimized transplant shock, recovered and survived. Prior to transplanting, plantlets were photomixotrophic, as indicated by low photosynthetic rates (A). During acclimatization, RWC, g _s, E, and A were significantly lower two days after transplanting. However, within 6 days after transplanting, plantlets recovered and became photoautotrophic – attaining high A, g _s, and E. Water use efficiency was initially low during the first days after transplanting, but increased dramatically at the end of the acclimatization period in part due to increased A. The stabilization and improvement of plantlet water status and gas exchange during acclimatization and post-acclimatization closely correlated with increased plantlet growth. This revised version was published online in June 2006 with corrections to the Cover Date.     

Arbuscular mycorrhizal fungi influence water relations, gas exchange, abscisic acid and growth of micropropagated chile ancho pepper (Capsicum annuum) plantlets during acclimatization and post-acclimatization

Little is known about the role of arbuscular mycorrhiza fungi (AMF) on physiological changes of micropropagated plantlets during acclimatization and post-acclimatization. Using chile ancho pepper (Capsicum annuum L. cv. San Luis), measurements were made of water relations, gas exchange, abscisic acid (ABA), plantlet growth and AMF development. Plantlets had low photosynthetic rates (A) and poor initial growth during acclimatization. Relative water content (RWC) decreased during the first days after transfer from tissue culture containers to ex vitro conditions. Consequently, transpiration rates (E) and stomatal conductance (gs) declined, confirming that in vitro formed stomata were functional and able to respond ex vitro to partial desiccation--thus avoiding excessive leaf dehydration and plant death. Colonization by AMF occurred within 3 days after inoculation. Colonized plantlets had lower leaf ABA and higher RWC than noncolonized (NonAMF) plantlets during peak plant dehydration (6 days after plant transfer)--and a higher A and gs as early as days 5 and 7. During post-acclimatization [after day 8, when RWC increased and stabilized], A increased in all plantlets; however, more dramatic changes occurred with AMF plantlets. Within 48 days, 45% of the roots sampled of inoculated plantlets were colonized and had extensive arbuscule development. At this time, AMF plantlets also had greater E, A, leaf chlorophyll, leaf elemental N, P and K, leaf dry biomass and leaf area, fruit production and differences in carbon partitioning [lower root/shoot ratio and higher leaf area ratio] compared with NonAMF plantlets. Rapid AMF colonization enhanced physiological adjustments, which helped plantlets recover rapidly during acclimatization and obtain greater growth during post-acclimatization.     

Field performance of micropropagated,macropropagated, and seed-derived propagules of three Eucalyptus grandis ortets

Tree size, survival, and coppicing of micropropagated plantlets, macropropagated cuttings, and seedlings of Eucalyptus grandis Hill ex Maiden were monitored through 57 months in a study in southern Florida to assess propagation options. Two plantlet lines developed by direct micropropagation and orchard open-pollinated seedlings from three ortets were compared in the main study. Rooted cuttings from up to four ramets of each of the three ortets and another ortet were examined in an adjacent supplemental study. Freezes at six and 16 months killed most initial and first-coppice stems to the ground. Most developmental differences in the main study were consistent from ages 2 to 57 months. Propagation by ortet interactions were observed beginning at 21 months, due to the poor performance of seedlings of one ortet after the second freeze. At 57 months, no differences in tree height, DBH, volume, or survival were detected between plantlet lines and between rooted cuttings and plantlets, but seedlings were inferior to plantlets and cuttings. Vegetative propagules had more uniform tree size at every age, with typically less than one-half the variability observed among seedlings. Even though plantlets and cuttings may be more expensive to produce, they have numerous advantages over seedlings for E. grandis plantation establishment in Florida.     

Plantlet morphology and the regulation of net water loss in tissue-cultured Douglas-fir

Tissue-culture plantlets of Douglas-fir [ Pseudotsuga menziesii (Mirb.) Franco] were highly susceptible to detrimental water loss upon removal from culture in vitro. Control of net water loss was related to shoot and root morphology. Relative water content after 3 h of atmospheric water stress was positively correlated to root number, root surface area, and the length of the longest root, and was inversely correlated to the ratio of needle surface area/root surface area. High relative water content apparently was a result of a higher rate of water uptake among plantlets with beneficial morphological features. It is recommended that, to improve the ability of a plantlet to withstand water stress during acclimatization, beneficial root system features be focused upon during plantlet production.     

‘欧美杨107’组培苗瓶外生根

‘欧美杨107’是近年来重点推广的杨树品种之一,但其组培苗存在移栽成活率不高的问题。本试验研究了‘欧美杨107’组培苗的直接瓶外生根和瓶内复壮后瓶外生根两种生根方式,结果表明,两种方式蘸取IBA的最佳浓度分别为10mg·L-1和100mg·L-1,生根率分别达73.33%和81.67%,移栽成活率分别为62.5%和93.33%。进一步研究发现生根的过程中外源补加0.5mg·L-1IBA溶液能加速生根,使生根周期缩短2周左右。添加1/2MS营养液可促进生根苗茎的生长和根的伸长,有利于移栽成活。瓶内生根苗的生根率高达到95%,但是移栽难以成活,显微镜观察发现瓶外生根苗比瓶内生根苗的根毛发达,验证了瓶外生根苗的根吸收功能更好。该研究对促进‘欧美杨107’组培苗及其转基因苗的推广应用具有重要的现实意义。     

Acclimatizing micropropagated black cherry by comparison with half-sib seedlings

Black cherry ( Prunus serotina Ehrh.) plantlets were cultured in vitro, transferred to potting mixture and evaluated for 8 weeks for acclimatization to a varying, but controlled environment. Whole plantlet growth and water relations were monitored and compared to seedlings of comparable size of the same maternal genotype grown under different conditions, but given the same pretest environment. At one week ex vitro. gravimetrically determined leaf conductance of plantlets was high, but became closer to that of seedlings in both magnitude and diurnal pattern as acclimatization progressed. By 8 weeks, leaf conductance of plantlets was nearly identical to that of seedlings, but the xylem water potential of the plantlets was significantly less. Logarithmic regressions of shoot vs root dry weight indicated that seedlings were allocating twice as much dry matter to shoot than to root growth compared to 4- and 8-week plantlets. Over the same period, stomatal densities of both seedlings and plantlets decreased and stomatal pore lengths increased. Multiple adventitious roots of plantlets emerged from a single site just below the root collar whereas secondary or lateral roots of seedlings originated acropetally along the central root axis. Leaf conductance of plantlets at 8 weeks was similar to that of seedlings indicating satisfactory acclimatization. The larger relative root growth rate of plantlets compared to the shoot, however, was associated with lower stem xylem water potential. The anomaly may be a consequence of the method of in vitro root formation. Plantlet growth rates were lower than growth rates of seedlings and their leaf area was correspondingly less.     

Number of air exchanges,sucrose concentration,photosynthetic photon flux,and differences in photoperiod and dark period temperatures affect growth of Rehmannia glutinosa plantlets in vitro

Rehmannia glutinosa plantlets were cultured for 4 weeks under different culture conditions to determine the optimum environment for in vitro growth and ex vitro survival. Plantlet growth increased with an increasing number of air exchanges of the culture vessel, exhibiting greatest shoot weight, total fresh weight, leaf area, and chlorophyll content at 4.4 h⁻¹ of air exchanges. High sucrose concentration (30 g l⁻¹) increased root weight but reduced shoot growth. Net photosynthetic rates of the plantlets were greatest when sucrose was not added to the medium. On the other hand, ex vitro survival of the plantlets was not influenced by sucrose concentration. In the experiment on difference in photoperiod and dark period temperatures (DIF) and photosynthetic photon flux (PPF), plantlet growth increased as DIF and PPF levels increased. Particularly, increasing PPF level had a more distinctive effect on plantlet growth than increasing DIF level. The interaction of DIF × PPF was also significant, showing the greatest plantlet growth in positive DIF (+8 DIF) and a high PPF (210 μmol m⁻² s⁻¹). In conclusion, the results of this experiment suggest that increased number of air exchanges of the culture vessel, decreased sucrose concentration, and positive DIF in combination with high PPF level enhanced growth and acclimatization of Rehmannia glutinosa plantlets. This revised version was published online in June 2006 with corrections to the Cover Date.     

珠美海棠试管苗的土壤支撑生根培养和带坨移栽

以粘壤土做培养基支撑物,选用只含有0．5mg·L^-1IBA和15g·L^-1蔗糖的液体培养基,珠关海棠试管苗茎段生根培养的结果显示,试管苗生根率可达到100．0％,明显高于琼脂支撑培养,根长也显著提高,并且长出正常的根毛。土支撑培养生根的珠美海棠试管苗开瓶炼苗21d后带坨移入营养钵中,移栽后不喷雾、并毋需覆盖塑膜、午后空气相对湿度控制在50％-65％,其成活率达到92．2％;在开瓶炼苗21d后,黑暗中叶片气孔的关闭率从26．7％增加到88．5％。     

Enhancement of chilling resistance of inoculated grapevine plantlets with a plant growth-promoting rhizobacterium, Burkholderia phytofirmans strain PsJN

In vitro inoculation of Vitis vinifera L. cv. Chardonnay explants with a plant growth-promoting rhizobacterium, Burkholderia phytofirmans strain PsJN, increased grapevine growth and physiological activity at a low temperature. There was a relationship between endophytic bacterial colonization of the grapevine plantlets and their growth at both ambient (26 degrees C) and low (4 degrees C) temperatures and their sensitivities to chilling. The major benefits of bacterization were observed on root growth (11.8- and 10.7-fold increases at 26 degrees C and 4 degrees C, respectively) and plantlet biomass (6- and 2.2-fold increases at 26 degrees C and 4 degrees C, respectively). The inoculation with PsJN also significantly improved plantlet cold tolerance compared to that of the nonbacterized control. In nonchilled plantlets, bacterization enhanced CO(2) fixation and O(2) evolution 1.3 and 2.2 times, respectively. The nonbacterized controls were more sensitive to exposure to low temperatures than were the bacterized plantlets, as indicated by several measured parameters. Moreover, relative to the noninoculated controls, bacterized plantlets had significantly increased levels of starch, proline, and phenolics. These increases correlated with the enhancement of cold tolerance of the grapevine plantlets. In summary, B. phytofirmans strain PsJN inoculation stimulates grapevine growth and improves its ability to withstand cold stress.     

In vitro and ex vitro rooting of Siratia grosvenorii, a traditional medicinal plant

Since a decade, the large-scale commercial production of Siratia grosvenorii plantlets is being practiced through in vitro culture of its microcuttings, but it has some drawbacks such as handling of plantlets, low transplant-survival rate, development of massive callus, low yield after transplantation, etc. An experiment has been conducted to improve the prevailing technique as well as to develop a new ex vitro technique to overcome these drawbacks. Several concentrations of naphthalene acetic acid (NAA) (0–4.0 mg/l) have been tried with the MS (Murashige and Skoog in Physiol Plant 15:473–479, 1962) basal medium containing 3% (w/v) sucrose and 4.0 g/l agar, out of which 0.1 mg/l NAA was found best in terms of smaller diameter of callus and maximum rooting and transplant survival rate. Further, use of perlite instead of agar medium also showed possibilities for future research on commercial-scale plantlet production. Ex vitro rooting technique was found superior to the in vitro one as plantlets developed through this method had lateral roots without any callus at the base of microcuttings, just like the natural root system and of course with higher root length, rooting rates, and transplant survival rate compared to the in vitro developed plantlets. Further, this technique is economical in terms of labor and time saving and gives rise to vigorous plants which ultimately bring higher yields and profits.     

香蕉离体茎尖超低温保存研究

以香蕉(Musaspp.)试管苗为试材,对其离体茎尖小滴玻璃化法超低温保存的影响因素进行了研究。小滴玻璃化法和玻璃化法超低温保存后再生率的差异表明,香蕉更适合用小滴玻璃化法进行超低温保存。香蕉小滴玻璃化法超低温保存的方案如下:试管苗在60g/L蔗糖的MS培养基上培养1~2个月,剥离带有1~2片叶原基的茎尖,室温下装载30m in(可延长至4h),0℃下PVS2处理40~50m in。6个基因型的14个品种的再生率平均为46.9%。通过SSR分子标记检测,再生植株的遗传稳定性没有发生改变。该结果为香蕉种质资源的长期保存提供了理论依据和技术支撑。     

Scaling-up production and field performance of micropropagated medicinal herb ‘Safed Musli’ (<Emphasis Type="Italic">Chlorophytum borivilianum</Emphasis>)

Summary Chlorophytum borivilianum Sant. et Fernand. (‘Safed Musli’), an endangered Indian medicinal herb, is valued for its tuberous roots reputed to have aphrodisiac properties. Farmers in India cultivate this medicinal herb on a commercial scale because of its high economic value. To cater to the growing demand for planting material, a highly reproducible field-tested and cost-effective micropropagation scheme has been developed. Best shoot multiplication was achieved on agargelled Murashige and Skoog medium containing 22.2 μM 6-benzylaminopurine (BA) and 3% sucrose. Phytagel^™ at 0.2% showed slightly better response than BDH agar in terms of shoot multiplication, but the use of BDH agar was preferred due to its low cost. With the optimized conditions, more than 15 000 plantlets could be produced in 20 wk. Plantlets subjected to hardening under agro-shadenet conditions during the monsoon months of high humidity showed better survival rate and growth compared to plantlets hardened in vitro and subsequently transferred to the greenhouse for acclimatization. Rate of plantlet survival was 87 and 90% under open field and agro-shadenet conditions, respectively. Plantlets grown ex vitro under agro-shadenet and field conditions produced tuberous roots which could be grown in the next season as a secondary propagule. We concluded that in vitro production of ‘Safed Musli’ was cost-effective compared to conventional propagation and holds great potential for commercial production.     

AgNO3对网纹甜瓜试管苗糖代谢及抗氧化酶活性的影响

在网纹甜瓜继代培养过程中加入不同浓度的AgNO3,测定试管苗糖代谢及抗氧化酶活性等。结果表明,玻璃苗果糖、葡萄糖和可溶性糖含量显著降低,而蔗糖含量显著高于正常苗;抗氧化系统发生紊乱,O2产生速率降低,H2O2含量下降,MDA含量增加,木质素含量降低。加入30μmol·L^-1AgNO,使玻璃苗蔗糖合成酶、蔗糖转化酶活性和木质素含量与正常苗差异不显著。说明适当浓度AgNO3有利于维持试管苗糖代谢正常进行,降低试管苗脂质过氧化程度,从而降低或抑制玻璃化现象的发生。     

Enhancement of Chilling Resistance of Inoculated Grapevine Plantlets with a Plant Growth-Promoting Rhizobacterium, Burkholderia phytofirmans Strain PsJN

In vitro inoculation of Vitis vinifera L. cv. Chardonnay explants with a plant growth-promoting rhizobacterium, Burkholderia phytofirmans strain PsJN, increased grapevine growth and physiological activity at a low temperature. There was a relationship between endophytic bacterial colonization of the grapevine plantlets and their growth at both ambient (26°C) and low (4°C) temperatures and their sensitivities to chilling. The major benefits of bacterization were observed on root growth (11.8- and 10.7-fold increases at 26°C and 4°C, respectively) and plantlet biomass (6- and 2.2-fold increases at 26°C and 4°C, respectively). The inoculation with PsJN also significantly improved plantlet cold tolerance compared to that of the nonbacterized control. In nonchilled plantlets, bacterization enhanced CO₂ fixation and O₂ evolution 1.3 and 2.2 times, respectively. The nonbacterized controls were more sensitive to exposure to low temperatures than were the bacterized plantlets, as indicated by several measured parameters. Moreover, relative to the noninoculated controls, bacterized plantlets had significantly increased levels of starch, proline, and phenolics. These increases correlated with the enhancement of cold tolerance of the grapevine plantlets. In summary, B. phytofirmans strain PsJN inoculation stimulates grapevine growth and improves its ability to withstand cold stress.     

Cryopreservation of porcine embryos by vitrification: A study of in vitro development

Until recently, attempts to preserve porcine embryos have been unsuccessful. Vitrification has been developed as a method of cryopreserving mammalian embryos by avoiding ice crystal formation, assuring a cryopreserved glass state during storage in liquid nitrogen. Vitrification may be a useful method of overcoming the deleterious effects of chilling injury when pig embryos are cryopreserved using conventional slow freezing procedures. In this study, we applied vitrification procedures for rodent and/or bovine embryos to cryopreserve porcine embryos. Following warming, survival was defined as normal development of embryos in culture, namely the formation or reexpansion of the blastocoelic cavity. Experiment 1 tested the relative toxicity of 3 vitrification procedures on Day-5, 6 and 7 porcine embryos. Embryos equilibrated in vitrification solution (VS3a) continued to develop in vitro at rates comparable to that of untreated control embryos. Experiment 2 was designed to evaluate embryonic development following cryopreservation by vitrification in VS3a. Day-5 porcine embryos did not survive cryopreservation while Day-6 and Day-7 embryos survived and continued development in vitro. In Experiment 3, we evaluated a period of culture prior to vitrification and its effect on cryosurvivability of porcine embryos. A 3-h culture period prior to vitrification had no effect on cryosurvivability over that of freshly recovered, immediately vitrified embryos. These studies indicate, for the first time, that porcine embryos can be successfully cryopreserved by vitrification based on morphology and subsequent development in vitro. However, survival following cryopreservation appears to depend upon embryonic age or stage of development.     

Regulation of growth of Lilium plantlets in liquid medium by application of paclobutrazol or ancymidol, for its amenability in a bioreactor system: growth parameters

Effect of growth retardants (paclobutrazol or ancymidol) was studied in Lilium plantlets growing in liquid culture. A significant increase in leaf chlorophyll, epicuticular wax, plant dry weight and bulb starch contents were found in plantlets treated with growth retardants. A similar increase in the number of leaves, roots and bulbs was also noted. However, total leaf area and the fresh weight increased only marginally. These features resulted in robust plantlets that showed significantly improved ex vitro survival. Based on these features, a comprehensive index (CI) was calculated as a measure of quality of the plantlets, and it correlated well with their ex vitro survival. Treatment of plantlets with 3.4 μM paclobutrazol was found to be the best and its carry over effects were also minimal.