Pineapple (Ananas comosuss, var. Smooth cayenne), which is a popular tropical fruit, is propagated vegetatively. Con ventional propagation alone does not provide clean and adequate planting material demanded in Ethiopia. Recently, in vitro multiplication has become a promising technique for large-scale production. However, the acclimatization to the external environment procedure impedes the efficiency, which needs carefully optimized acclimatization techniques. We report optimized acclimatization procedures following first- and second-stage hardening methods for in vitro pineapple plantlets. Primarily, Jiffy-7 peat pellet allowed growing plants vigorously and provided above 8% survival rate over soil mix. Nevertheless, in Ethiopia, soil mix is cheaper and locally accessible. The primarily acclimatized plantlets are needed to be hardened further for better establishment and survival in the field. Black polybag and polysleeve pots filled with soil mix were evaluated in the greenhouse. A significant difference was obtained between pots for number of roots and substrate weight. Polybags had higher root number than polysleeves and saved about 27% of substrates per plant, which is a reduction of 25% of total transportation cost. Hence, the soil mix and polybags were found to be preferable over substrates and pots, for subsequent in vitro pineapple acclimatization.
Pineapple (Ananas comosuss L.) is one of the most popular and delicious triploid fruit. It is esteemed for its pronounced flavor and nutritive elements. Pineapple is propagated vegetatively through suckers, slips or crowns [
In Ethiopia, the major pineapple production sites are located in the southern and southwestern part of the country owned by private farmers and state farm. The farmers produce in small-scale on fragments of lands, whereas the state farm of Coffee Plantation Development Enterprise produces pineapple var. smooth cayenne along with their coffee and/or maize plantation [
In vitro propagation is a crucial technique for disease free, rapid, uniform and mass production of pineapple plantlets [3-5]. Nevertheless, ultimate success of in vitro produced plantlets depends upon the successful transfer and establishment of plants in ex vitro conditions. High loss or damage of in vitro raised plants can occurred when transferred to ex vitro conditions because of the transfer shock [
The successful establishment of in vitro raised plants on the soil and later on field is the major success of in vitro propagation [3,5,6]. Thus, in vitro pineapple plantlets need to be acclimatized carefully. The acclimatization period varies from 6 - 8 months until the plants reach an appropriate size (200 - 300 mm height) for field transfer [
Large-scale in vitro pineapple plantlets production has been started in Ethiopia a few years ago [
On the other hand, acclimatized plants do not have the appropriate size for direct planting into the field, which results in poor survival rate [
Pineapple (Ananas comosuss var. Smooth cayenne) plantlets were multiplied under in vitro condition according to the protocol established previously [4,11] in the plant biotechnology laboratory, Jimma Agricultural Research Center, Ethiopia. Well elongated, rooted and uniform plantlets were selected and taken out from culture Jam Jars (
The media were prepared using full strength Murashige and Skoog (MS) (1962) basal salts amended 3% (w/v) table sugar (local shop, Jimma, Ethiopia) and 2 mg/l BA for multiplication. Half strength MS medium supplemented with 3 mg/l IBA and 3% (w/v) table sugar
was also prepared for rooting. The pH of the medium was adjusted to 5.8 using 1 N NaOH or 0.1 N HCl. The media were then solidified with 0.8% (w/v) agar (Sigma Chemical Co. Germany) for multiplication and with composite of enset starch (local shop, Jimma, Ethiopia) (60 g/l) and agar (2 g/l) for rooting [
Two acclimatization media treatments were arranged in the greenhouse prior to planting. This was replicated 10 times. The first treatment medium was soil mix composing of soil, coffee husk and sand in 1:2:1 ratio, respectively according previous report [
In this experiment, two poly pot types such as black polysleeves (8 cm diameter) and polybags (8 cm diameter) were employed. The pots were easily accessed from a local market. Small holes were made at the bottom of polybags using a paper punch to allow aeration and drainage of excess water. The pots were filled with soil mix, which was similar substrate as first acclimatization treatments. The filling was done by hand with different pressure intensities, a bit compact for polysleeves to avoid loss of soil at the bottom and a loose filling for polybags. Then, pots were arranged properly and placed on the flour of the greenhouse. Later, the plants acclimatized during first-stage acclimatization were transplanted into both pot types and kept on the greenhouse floor (
Non-destructive and destructive data were collected from first and second stage-acclimatization after 2.5 and 4 months, respectively. Non-destructive parameters collected were survival rate (%), number of leaves and roots, length of shoot and root (cm) as well as girth (mm). On the other hand, fresh and dry weight of shoot and root (g) as well as soil weight (g) were collected through destructive method. Fresh weight was measured immediately after removing the soil and separating shoots from roots. Dry matter of shoots and roots were taken after drying in an oven at 70˚C for 48 h following procedures [
In vitro multiplied pineapple plantlets need to be acclimatized in the greenhouse prior to transferring into the field. Since acclimatization of in vitro plantlets in ex vitro conditions is a critical step for survival and growth performance, two major hardening stages have been evaluated.
In vitro raised pineapple plantlets were planted into seedling trays filled with locally prepared soil mix and commercially available Jiffy-7 pellets. The survival rate and growth performance of plantlets were evaluated. The result revealed that plantlets grown in Jiffy-7 peat pellet showed above 98% of survival followed by soil mix (1 soil, 2 coffee husk and 1 sand) with above 90% of survival (
Because of the small size and weakness of the first-stage acclimatized pineapple plantlets, further hardening of these plantlets is required to increase the strength and growth performance. After transplanting those plantlets into different pot types, the plant performance and costs of substrates and transportation were evaluated. A significant difference (p < 0.05) for number of root was observed between pot types. Higher mean root number (19.22) was obtained from polybags than polysleeves (16.11) (
Highly significant difference (p < 0.01) for the amount of substrates (1 soil, 2 coffee husk and 1 sand) in each pot types was observed during this experiment. The highest mean value (1225.05 g) were measured from polysleeves in contrary to polybags that had a mean value of 892.14 g. Using polybag for this stage of acclimatization reduced about 27% of the substrates needed per plant compared to polysleeves (
along with substrate costs reduction over polysleeves. Evidently, replacing polysleeves with polybags, transportation cost of potting plants into growing field is reduced by about 25 percent.
Means followed by the same letter within the same column are not significantly different. All growth parameters per substrate are mentioned as mean value. No. = number, Wt = weight, = (±SE) standard error, CV = coefficient of variation, NS = Non-significant; * = significant at p < 0.05; ** = highly significant at p < 0.01.
Means followed by the same letter within the same column are not significantly different at 0.05 probability level. All growth parameters are as mean value/plant. No. = Number, Wt = weight, = ± (SE) standard error, * = significant different; ** = highly significant different.
Pineapple (Ananas comosuss var. Smooth cayenne) is one of the vegetatively propagated tropical fruit crops. In vitro propagation is implemented widely for large-scale pineapple planting materials production. Acclimatization or hardening is a crucial step prior to transplantation of plantlets into the field. Pineapple requires a long acclimatization period until it reaches an appropriate size [
In vitro pineapple plantlets were primarily acclimatized on different substrates. As the result showed, the substrates influenced the survival rate in the greenhouse, which was about 90% in soil mix and 98% in commercially available substrate called Jiffy-7 peat pellet. This result shows that the survival rate on soil mix was improved by 5% as compared to previous report [
It is suggested that modified soil mixture, which has more organic matter and equal proportion of soil and sand, improves aeration and reduces water retention leading to root growth, which comprises improved root and shoot-root connection for hydraulic conductivity [
In contrast, Jiffy-7 peat pellet showed greatest survival rate and good plant performance, suggests that its relatively low pH facilitates nutrient uptake [8,17] and its high water holding capacity [
Since pineapple growing fields are located throughout the country, the plantlets should be strong, vigorous, well acclimatized and simple for handling to tolerate transportation and field shocks [
Different types of pots are employed for growing plants in the nursery or the greenhouse. Similar to media types, the pot type and size affect the plant growth performance [
Two subsequent hardening stages for in vitro raised pineapple plantlets were optimized prior to transferring them into the field for Ethiopian condition. Soil mix (1 soil, 2 coffee husk and 1 sand) was found as a cheap and locally available material, making it a good substrate for primary acclimatization in the greenhouse although Jiffy-7 peat pellet improved growth performance and survival rate slightly more. Using polybags was also the preferable pot in terms of cost and usefulness to obtain a better plantlet performance during secondary acclimatization.
The authors would like to appreciate all staff of Plant biotechnology research laboratory, Jimma Agricultural Research Center for their unreserved contribution of the work. Special gratitude goes to Mr. Zakir Abbanega for his technical help throughout the experiments and to Ms. Roman Getachew for her contribution during data collection. Our deep gratitude goes to the German Centre for International Migration and Development (CIM) which supported the expert in the work. We also thank European Union and MASHAV-USAID that donated Jiffy-7 peat pellets along with seedling trays and support the work respectively.