Plant Cell Reports
Plant Cell Reports (1992) 11:532-534
9 Springer-Verlag 1992
Callus induction and adventitious organogenesis of kenaf (Hibiscus cannabinus L.) K.S. McLean 1, G.W. Lawrence 2, and N . A . Reichert 3 1 D e p a r t m e n t o f Agriculture, N o r t h e a s t L o u i s i a n a U n i v e r s i t y , M o n r o e , L A 71209, U S A 2 D e p a r t m e n t o f P l a n t P a t h o l o g y a n d Weed Science a n d 3 D e p a r t m e n t o f H o r t i c u l t u r e , Mississippi State University, Mississippi State, M S 39762, U S A Received N o v e m b e r 20, 1991/Revised v e r s i o n received J u n e 23, 1992 - C o m m u n i c a t e d by G . C . Phillips
Abstract. Callus production along with caulogenesis and rhizogenesis were obtained from internodal stem explants of kenaf (Hibiscus cannabinus L.) after 4 weeks in culture. Murashige and Skoog medium was used for two 4 X 4 matrix experiments designed to determine suitable growth regulator combinations (NAA/BAP or 2,4-D/kinetin) and concentrations (0.1, 0.3, 1.0, 3.0 mg/L). The most abundant callus production was observed at 0.3/3.0 and 1.0/3.0 mg/L 2,4-D/kinetin and at 1.0/1.0 and 3.0/1.0 mg/L NAA/BAP. Rhizogenesis was most extensive with NAA/BAP at concentrations of 0.1/3.0 and 0.3/3.0 mg/L. Adventitious shoots developed on both auxin/cytokinin matrixes when each concentration was at 0.3 mg/L or less. These protocols will facilitate the development of in vitro approaches to kenaf improvement and the study of certain hostpathogen interactions. Abbreviations: MS, Murashige and Skoog (1962) medium; 2,4-D, 2,4dichlorophenoxyacetic acid; BAP, 6-benzylaminopurine; NAA, 1-naphthyleneacetic acid; SDS, sodium dodecyl sulfate. Introduction Kenaf (Hibiscus cannabinus L.) is an annual fiber crop native to the tropical regions of Africa and the East Indies (Sij 1987). In Mississippi, it is currently being evaluated as a renewable source of pulp for paper production in the United States. Kenaf is an appealing alternative fiber source, either used alone or in combination with wood pulp, because it can be grown as an annual crop and thus the massive deforestation required to fulfill paper product demand could be reduced. Climatic conditions suitable for cotton (Gossypium hirsutum L.) production, found in much of the southern U.S., would be ideal for kenaf. Both are members of the Malvaceae and share susceptibility to many of the same pathogens, including parasitic nematodes (Lawrence and McLean 1991). The development of in vitro gnotobiotic systems, along with plant regeneration capabilities, would be advantageous for Correspondence to: G . W . L a w r e n c e
analysis of plant host-parasite interactions and plant improvement. Reports on in vitro manipulation of Hibiscus spp. have been limited. Roselle (Hibiscus sabdar~..ffa L.) callus has been induced from seedling tissue and maintained in vitro (Mizukami et al. 1988). Callus and somatic embryogenesis were induced from hypocotyl tissue (Reynolds et al. 1981). To our knowledge, ours is the first report on in vitro studies of kenaf. We achieved callus growth and/or organogenesis induced on internodal stem sections of kenaf and have determined suitable auxin/cytokinin concentrations for such growth. Materials and Methods Murashige and Skoog (1962) basal salts and vitamins (MS) with 100 mg/L myo-inositol, 30 g/L sucrose, 8 g/L Phytoagar, pH 5.8 served as the basic medium (MS-O~ of the matrixes. MS-O was sterilized in the autoclave at 1.05 kg/cm z pressure for 25 min (121~ Plant growth regulators were filter sterilized with 0.2 [axn filters and added to cooled MS-O. Auxin/cytokinin combinations tested were NAA/BAP and 2,4-D/kinetin each added at final concentrations of 0.1, 0.3, 1.0, or 3.0 mg/L in all possible combinations (in two 4 x 4 matrix experiments). MS-O served as the negative control. Stems of kenaf, variety Tainung 1, were selected from four month old plants grown on the North Plant Science Research Farm at Mississippi State University. A 15 cm stem section was removed from the center of each 2.4 m stem. Sections were Surface sterilized for 15 min in a 0.5% sodium hypochlorite plus 0.1% SDS solution followed by three 5 min rinses in sterile distilled water. Intemodal stem sections were aseptically trimmed into 0.5 cm lengths, sprit longitudinally, and randomly placed 5 per 100 x 25 mm petri plate, split side down, on each of the 33 different media. All plates were incubated under a 16 h right: 8 h dark photoperiod at 27~ for four weeks. A completely randomized design with three replications per medium treatment was used. Stem sections were incubated at 27~ for a total of four weeks. The experiment was repeated once. Kenaf stem cultures were rated at weekly intervals. To set numerical values that represented both qualitative and quantitative growth, a rating scale from 0 to 9 was developed. The scale was defined as: 0 - no tissue growth, 1 - initial callus growth from stem ends, 2 - callus arising from one stem end, 3 - callus arising from both stem ends, 4 - callus arising from both stem ends and the epidermis, 5 - callus arising from both stems ends, epidermis and underlying parenchyma tissue, 6 - callus formation encompassing the stem section, 7 - callus growth double the original estimated stem section mass, 8 - callus growth 3 times the original estimated mass, and 9 - callus growth 4 times the original estimated mass. Rhizogenesis or caulogenesis was recorded from tissue sections at 4 weeks. Data w e r e analyzed by ANOVA and means separated by Fisher's protected least significant difference test. Roots were excised from stem explants and cultured on the same media from which they originated. Woody Plant Medium (Lloyd and McCown 1980) supplemented with 140 mg/L myo-inositol, 10 g/L
533 dextrose, 5 g/L Phytoagar, and 0.57 mg/L indole-3-acetic acid and Murashige Syngonium Stage I]1 Pretransplant Medium (Miller and Murashige 1976) were used for root initiation on adventitious shoots. Rooted shoots were then subsequently transplanted into a peat-sand medium (2:1 v/v), and grown in a controlled environmental chamber. All transplants were incubated under a 16 h light: 8 h dark photoperiod at 27~ as previously described.
Results and Discussion
MS-O supplemented with NAA/BAP and 2,4-D/kinetin served as effective media for callus production in kenaf. Ninety-five percent of the stem sections began producing callus on all media except MS-O after 5 d culture. Callus protruded through the epidermis of the stem and from parenchyma tissue exposed on the split stem surface. Overall, callus production was greatest on the 2,4-D/ kinetin matrix. Callus morphology also differed. Dense, green pigmented callus was produced on the NAA/BAP GROWTH
O
INDEX
respectively, of stem sections producing roots (Fig.3). Roots obtained lengths in excess of 2 cm and included extensive branching and root hair growth. The 2,4-D/ kinetin matrix produced limited rhizogenesis at all concentrations, but was much less abundant than the rhizogenesis on the NAA/BAP media. Four weeks after initiation, limited adventive caulogenesis was observed developing from callus in both matrixes. Adventitious shoots were produced with a frequency of 3, 6, 6, and 13%, respectively, on MS-O media supplemented with 0.1 mg/L 2,4-D, 0.1 mg/L kinetin; 0.3 mg/L 2, 4-D, 0.1 mg/L kinetin; 0.1 mg/L 2,4-D, 0.3 mg/L kinetin; and 0.3 mg/L 2,4-D, 0.3 mg/L kinetin (Table 1). Shoots were produced with a frequency of 6, 6, and 3%, respectively, on MS-O media supplemented with 0.1 mg/L NAA, 0.1 mg/L BAP; 0.3 mg/L NAA, 0.1 mg/L BAP; and 0.1 mg/L NAA, 0.3 mg/L kinetin. Adventitious caulogenesis was not observed on the other auxin/cytokinin concentrations of both matrixes. Adventitious shoots placed on either the Woody Plant Medium or Murashige Syngnoium Stage III Pretransplant Medium were equally successful in producing roots. An average of 83% of the adventitious shoots produced roots when placed on either media. Rooted shoots were transplanted into a peat-sand medium and maintained in an environmental growth chamber (Fig. 4). The frequency of survival of all transplants was 38% without prior hardening.
........................................... !i1
8
6
' N I @ I1N IIN f
'~
GROWTH
INDEX
.............................. 0.1
0.3
1.0
0
'2
a.o
NAA/BAP mg/L Fig.1. Growth scale rating of kenaf callus on NAA/BAP matrix after 4 weeks in culture. NAA concentrations are represented on the X axis and BAP concentrations are presented on the Z axis. Data represent the means of 30 stern pieces per medium. Means compared using Fisher's protected least significant difference test. (See Materials and Methods for description of growth scales.)
matrix compared to the yellow-white, friable callus produced on the 2,4-D/kinetin matrix. Media browning, most likely due to oxidized polyphenols, appeared in NAA/BAP plates two weeks after plating. Browning was not associated with any particular concentration or ratio of NAA and BAP, and was less apparent when the experiment was repeated (with donor tissue in the reproductive stage). No 2,4-D/kinetin combination induced browning. After four weeks, all 2,4-D/kinetin combinations produced more callus overall, but two NAA/BAP combinations also yielded large amounts. Within the NAA/BAP matrix, greatest callus production was on 1.0/1.0 and 3.0/1.0 mg/L plates with growth ratings of 7.8 and 8.3, respectively (Fig.l). The best 2,4-D/kinetin combinations for callus production were 0.1/3.0, 0.3/3.0, and 1.0/3.0 mg/L with growth ratings of 7.8, 8.0, and 7.8, respectively (Fig.2). A very small amount of callus tissue was produced on MS-O after 4 weeks in culture, but no organogenesis was observed. Adventitious rhizogenesis was induced on all NAA/BAP combinations, but was most extensive on 0.1/3.0, 0.3/3.0, and 0.3/1.0 mg/L with 90, 86, and 86%,
.0
O
V 0.1
~
/
~ 0.3
/
~
/ 1.0
~
/
O. 1
8.0
2,4-D/KINETIN mg/L Fig.2. Growth scale rating of kenaf callus 2,4-D/kinetin matrix after 4 weeks in culture. 2,4-D concentrations are represented on the X axis and kinetin concentrations are presented on the Z axis. Data represent the means of 30 stem pieces per medium. Means compared using Fisher's protected least significant difference test. (See Materials and Methods for description of growth scales.)
In summary, both hormone combinations at various concentrations produced an abundance of kenaf callus. Callus has been continuously cultured on these media for 1 year. Callus growth appeared earlier on NAA/BAP plates, although was less abundant than that on 2,4-D/ kinetin plates. Media browning did not occur in the 2,4-D/kinetin matrix medium. The most vigorous roots (on NAA/BAP) were cut from the stem explants and continuously cultured on the same media from which they originated for a total of 1 year. In vitro studies of plant colonization and resistance screening using obligate
534 parasitic pathogens is possible via callus and root culture (Msikita et al. 1988; Pollard and Walker 1990). We have determined suitable hormone combinations and concentrations for achieving callus and root induction and maintenance for kenaf. Although this was the main emphasis as a precursor to subsequent development and study of certain plant-pathogen interaction systems, shoot induction was also observed and whole plants were obtained. It was important to discover that kenaf internodal stem sections are capable of generating
adventitious shoots because certain plant improvement strategies require the recovery of whole plants from cell cultures manipulated in vitro. PERCENT
ROOT P R O D U C T I O N
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Percent shoot organogenesis produced from 4 week old kenaf callus.
Table
60
Auxin/Cytokinin concentration (mg/L)a
....
~ .
% Freouencv of Shoots 2,4-D/Kinetin NAA/BAP
0.1[0.1 0.3/0.1 0.1/0.3 0.3/0.3
3 6 6 13
6 6 3 0
LSD (P=0.05)
1.4
0.5
a Shoot organogenesis was not observed for all other media combinations.
!i!i!i:i:~:!:~:i
0.1
0.3
1.0
1.0
3.0
NAA/BAP mg/L Fig.3. Percent root production on NAA/BAP matrix from kenaf after 4 weeks in culture. NAA concentrations are represented on the X axis and BAP concentrations are presented on the Z axis. Data represent the means of 30 stem pieces per medium. Means compared using Fisher's protected least significant difference test.
References Donovan A, Isacc S, and CoUin HA (1990) In: Pollard JW, Walker JM (eds) Plant cell and tissue culture. Humaan Press, Clifton New Jersey, pp 405-412 Lawrence GW, McLean KS (1991) Mississippi Agric & For Exper Sta Res Rep Vol 16, No 2 Lloyd G, McCown B (1980) Proc Int Plant Prop Soc 30:421427 Miller LR, Murashige T (1976) In Vitro 12:796 Mizukami H, Kaomi T, Ohashi H, Noboru H (1988) Plant Cell Rep 7:553-556 Msikita W, Wilkinson HT, Skirvin RM (1990) Hort Sci 25:967-969 Murashige T, Skoog F (1962) Physiol Plant 15:473-497 Reynolds BD, Blackmon WJ, Postek CE (1981) Environ Expt Bot 21:438 Sij JW (1987) Texas Agric Exper Stat Rep PR-4536
Fig.4. Adventitious shoot induction from 4 week old kenaf callus. (A) Shoot initial formation from callus; (B) Multiple shoot formation from callus; (C) Root formation from shoots; and (D) Regenerated plantlet growing in a peat medium.
Plant Cell Reports (1992) 11:532-534
9 Springer-Verlag 1992
Callus induction and adventitious organogenesis of kenaf (Hibiscus cannabinus L.) K.S. McLean 1, G.W. Lawrence 2, and N . A . Reichert 3 1 D e p a r t m e n t o f Agriculture, N o r t h e a s t L o u i s i a n a U n i v e r s i t y , M o n r o e , L A 71209, U S A 2 D e p a r t m e n t o f P l a n t P a t h o l o g y a n d Weed Science a n d 3 D e p a r t m e n t o f H o r t i c u l t u r e , Mississippi State University, Mississippi State, M S 39762, U S A Received N o v e m b e r 20, 1991/Revised v e r s i o n received J u n e 23, 1992 - C o m m u n i c a t e d by G . C . Phillips
Abstract. Callus production along with caulogenesis and rhizogenesis were obtained from internodal stem explants of kenaf (Hibiscus cannabinus L.) after 4 weeks in culture. Murashige and Skoog medium was used for two 4 X 4 matrix experiments designed to determine suitable growth regulator combinations (NAA/BAP or 2,4-D/kinetin) and concentrations (0.1, 0.3, 1.0, 3.0 mg/L). The most abundant callus production was observed at 0.3/3.0 and 1.0/3.0 mg/L 2,4-D/kinetin and at 1.0/1.0 and 3.0/1.0 mg/L NAA/BAP. Rhizogenesis was most extensive with NAA/BAP at concentrations of 0.1/3.0 and 0.3/3.0 mg/L. Adventitious shoots developed on both auxin/cytokinin matrixes when each concentration was at 0.3 mg/L or less. These protocols will facilitate the development of in vitro approaches to kenaf improvement and the study of certain hostpathogen interactions. Abbreviations: MS, Murashige and Skoog (1962) medium; 2,4-D, 2,4dichlorophenoxyacetic acid; BAP, 6-benzylaminopurine; NAA, 1-naphthyleneacetic acid; SDS, sodium dodecyl sulfate. Introduction Kenaf (Hibiscus cannabinus L.) is an annual fiber crop native to the tropical regions of Africa and the East Indies (Sij 1987). In Mississippi, it is currently being evaluated as a renewable source of pulp for paper production in the United States. Kenaf is an appealing alternative fiber source, either used alone or in combination with wood pulp, because it can be grown as an annual crop and thus the massive deforestation required to fulfill paper product demand could be reduced. Climatic conditions suitable for cotton (Gossypium hirsutum L.) production, found in much of the southern U.S., would be ideal for kenaf. Both are members of the Malvaceae and share susceptibility to many of the same pathogens, including parasitic nematodes (Lawrence and McLean 1991). The development of in vitro gnotobiotic systems, along with plant regeneration capabilities, would be advantageous for Correspondence to: G . W . L a w r e n c e
analysis of plant host-parasite interactions and plant improvement. Reports on in vitro manipulation of Hibiscus spp. have been limited. Roselle (Hibiscus sabdar~..ffa L.) callus has been induced from seedling tissue and maintained in vitro (Mizukami et al. 1988). Callus and somatic embryogenesis were induced from hypocotyl tissue (Reynolds et al. 1981). To our knowledge, ours is the first report on in vitro studies of kenaf. We achieved callus growth and/or organogenesis induced on internodal stem sections of kenaf and have determined suitable auxin/cytokinin concentrations for such growth. Materials and Methods Murashige and Skoog (1962) basal salts and vitamins (MS) with 100 mg/L myo-inositol, 30 g/L sucrose, 8 g/L Phytoagar, pH 5.8 served as the basic medium (MS-O~ of the matrixes. MS-O was sterilized in the autoclave at 1.05 kg/cm z pressure for 25 min (121~ Plant growth regulators were filter sterilized with 0.2 [axn filters and added to cooled MS-O. Auxin/cytokinin combinations tested were NAA/BAP and 2,4-D/kinetin each added at final concentrations of 0.1, 0.3, 1.0, or 3.0 mg/L in all possible combinations (in two 4 x 4 matrix experiments). MS-O served as the negative control. Stems of kenaf, variety Tainung 1, were selected from four month old plants grown on the North Plant Science Research Farm at Mississippi State University. A 15 cm stem section was removed from the center of each 2.4 m stem. Sections were Surface sterilized for 15 min in a 0.5% sodium hypochlorite plus 0.1% SDS solution followed by three 5 min rinses in sterile distilled water. Intemodal stem sections were aseptically trimmed into 0.5 cm lengths, sprit longitudinally, and randomly placed 5 per 100 x 25 mm petri plate, split side down, on each of the 33 different media. All plates were incubated under a 16 h right: 8 h dark photoperiod at 27~ for four weeks. A completely randomized design with three replications per medium treatment was used. Stem sections were incubated at 27~ for a total of four weeks. The experiment was repeated once. Kenaf stem cultures were rated at weekly intervals. To set numerical values that represented both qualitative and quantitative growth, a rating scale from 0 to 9 was developed. The scale was defined as: 0 - no tissue growth, 1 - initial callus growth from stem ends, 2 - callus arising from one stem end, 3 - callus arising from both stem ends, 4 - callus arising from both stem ends and the epidermis, 5 - callus arising from both stems ends, epidermis and underlying parenchyma tissue, 6 - callus formation encompassing the stem section, 7 - callus growth double the original estimated stem section mass, 8 - callus growth 3 times the original estimated mass, and 9 - callus growth 4 times the original estimated mass. Rhizogenesis or caulogenesis was recorded from tissue sections at 4 weeks. Data w e r e analyzed by ANOVA and means separated by Fisher's protected least significant difference test. Roots were excised from stem explants and cultured on the same media from which they originated. Woody Plant Medium (Lloyd and McCown 1980) supplemented with 140 mg/L myo-inositol, 10 g/L
533 dextrose, 5 g/L Phytoagar, and 0.57 mg/L indole-3-acetic acid and Murashige Syngonium Stage I]1 Pretransplant Medium (Miller and Murashige 1976) were used for root initiation on adventitious shoots. Rooted shoots were then subsequently transplanted into a peat-sand medium (2:1 v/v), and grown in a controlled environmental chamber. All transplants were incubated under a 16 h light: 8 h dark photoperiod at 27~ as previously described.
Results and Discussion
MS-O supplemented with NAA/BAP and 2,4-D/kinetin served as effective media for callus production in kenaf. Ninety-five percent of the stem sections began producing callus on all media except MS-O after 5 d culture. Callus protruded through the epidermis of the stem and from parenchyma tissue exposed on the split stem surface. Overall, callus production was greatest on the 2,4-D/ kinetin matrix. Callus morphology also differed. Dense, green pigmented callus was produced on the NAA/BAP GROWTH
O
INDEX
respectively, of stem sections producing roots (Fig.3). Roots obtained lengths in excess of 2 cm and included extensive branching and root hair growth. The 2,4-D/ kinetin matrix produced limited rhizogenesis at all concentrations, but was much less abundant than the rhizogenesis on the NAA/BAP media. Four weeks after initiation, limited adventive caulogenesis was observed developing from callus in both matrixes. Adventitious shoots were produced with a frequency of 3, 6, 6, and 13%, respectively, on MS-O media supplemented with 0.1 mg/L 2,4-D, 0.1 mg/L kinetin; 0.3 mg/L 2, 4-D, 0.1 mg/L kinetin; 0.1 mg/L 2,4-D, 0.3 mg/L kinetin; and 0.3 mg/L 2,4-D, 0.3 mg/L kinetin (Table 1). Shoots were produced with a frequency of 6, 6, and 3%, respectively, on MS-O media supplemented with 0.1 mg/L NAA, 0.1 mg/L BAP; 0.3 mg/L NAA, 0.1 mg/L BAP; and 0.1 mg/L NAA, 0.3 mg/L kinetin. Adventitious caulogenesis was not observed on the other auxin/cytokinin concentrations of both matrixes. Adventitious shoots placed on either the Woody Plant Medium or Murashige Syngnoium Stage III Pretransplant Medium were equally successful in producing roots. An average of 83% of the adventitious shoots produced roots when placed on either media. Rooted shoots were transplanted into a peat-sand medium and maintained in an environmental growth chamber (Fig. 4). The frequency of survival of all transplants was 38% without prior hardening.
........................................... !i1
8
6
' N I @ I1N IIN f
'~
GROWTH
INDEX
.............................. 0.1
0.3
1.0
0
'2
a.o
NAA/BAP mg/L Fig.1. Growth scale rating of kenaf callus on NAA/BAP matrix after 4 weeks in culture. NAA concentrations are represented on the X axis and BAP concentrations are presented on the Z axis. Data represent the means of 30 stern pieces per medium. Means compared using Fisher's protected least significant difference test. (See Materials and Methods for description of growth scales.)
matrix compared to the yellow-white, friable callus produced on the 2,4-D/kinetin matrix. Media browning, most likely due to oxidized polyphenols, appeared in NAA/BAP plates two weeks after plating. Browning was not associated with any particular concentration or ratio of NAA and BAP, and was less apparent when the experiment was repeated (with donor tissue in the reproductive stage). No 2,4-D/kinetin combination induced browning. After four weeks, all 2,4-D/kinetin combinations produced more callus overall, but two NAA/BAP combinations also yielded large amounts. Within the NAA/BAP matrix, greatest callus production was on 1.0/1.0 and 3.0/1.0 mg/L plates with growth ratings of 7.8 and 8.3, respectively (Fig.l). The best 2,4-D/kinetin combinations for callus production were 0.1/3.0, 0.3/3.0, and 1.0/3.0 mg/L with growth ratings of 7.8, 8.0, and 7.8, respectively (Fig.2). A very small amount of callus tissue was produced on MS-O after 4 weeks in culture, but no organogenesis was observed. Adventitious rhizogenesis was induced on all NAA/BAP combinations, but was most extensive on 0.1/3.0, 0.3/3.0, and 0.3/1.0 mg/L with 90, 86, and 86%,
.0
O
V 0.1
~
/
~ 0.3
/
~
/ 1.0
~
/
O. 1
8.0
2,4-D/KINETIN mg/L Fig.2. Growth scale rating of kenaf callus 2,4-D/kinetin matrix after 4 weeks in culture. 2,4-D concentrations are represented on the X axis and kinetin concentrations are presented on the Z axis. Data represent the means of 30 stem pieces per medium. Means compared using Fisher's protected least significant difference test. (See Materials and Methods for description of growth scales.)
In summary, both hormone combinations at various concentrations produced an abundance of kenaf callus. Callus has been continuously cultured on these media for 1 year. Callus growth appeared earlier on NAA/BAP plates, although was less abundant than that on 2,4-D/ kinetin plates. Media browning did not occur in the 2,4-D/kinetin matrix medium. The most vigorous roots (on NAA/BAP) were cut from the stem explants and continuously cultured on the same media from which they originated for a total of 1 year. In vitro studies of plant colonization and resistance screening using obligate
534 parasitic pathogens is possible via callus and root culture (Msikita et al. 1988; Pollard and Walker 1990). We have determined suitable hormone combinations and concentrations for achieving callus and root induction and maintenance for kenaf. Although this was the main emphasis as a precursor to subsequent development and study of certain plant-pathogen interaction systems, shoot induction was also observed and whole plants were obtained. It was important to discover that kenaf internodal stem sections are capable of generating
adventitious shoots because certain plant improvement strategies require the recovery of whole plants from cell cultures manipulated in vitro. PERCENT
ROOT P R O D U C T I O N
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Percent shoot organogenesis produced from 4 week old kenaf callus.
Table
60
Auxin/Cytokinin concentration (mg/L)a
....
~ .
% Freouencv of Shoots 2,4-D/Kinetin NAA/BAP
0.1[0.1 0.3/0.1 0.1/0.3 0.3/0.3
3 6 6 13
6 6 3 0
LSD (P=0.05)
1.4
0.5
a Shoot organogenesis was not observed for all other media combinations.
!i!i!i:i:~:!:~:i
0.1
0.3
1.0
1.0
3.0
NAA/BAP mg/L Fig.3. Percent root production on NAA/BAP matrix from kenaf after 4 weeks in culture. NAA concentrations are represented on the X axis and BAP concentrations are presented on the Z axis. Data represent the means of 30 stem pieces per medium. Means compared using Fisher's protected least significant difference test.
References Donovan A, Isacc S, and CoUin HA (1990) In: Pollard JW, Walker JM (eds) Plant cell and tissue culture. Humaan Press, Clifton New Jersey, pp 405-412 Lawrence GW, McLean KS (1991) Mississippi Agric & For Exper Sta Res Rep Vol 16, No 2 Lloyd G, McCown B (1980) Proc Int Plant Prop Soc 30:421427 Miller LR, Murashige T (1976) In Vitro 12:796 Mizukami H, Kaomi T, Ohashi H, Noboru H (1988) Plant Cell Rep 7:553-556 Msikita W, Wilkinson HT, Skirvin RM (1990) Hort Sci 25:967-969 Murashige T, Skoog F (1962) Physiol Plant 15:473-497 Reynolds BD, Blackmon WJ, Postek CE (1981) Environ Expt Bot 21:438 Sij JW (1987) Texas Agric Exper Stat Rep PR-4536
Fig.4. Adventitious shoot induction from 4 week old kenaf callus. (A) Shoot initial formation from callus; (B) Multiple shoot formation from callus; (C) Root formation from shoots; and (D) Regenerated plantlet growing in a peat medium.
