Table of Contents
Reichert, N.A. and B.S. Baldwin. 1996. Potential for
kenaf improvement via somaclonal variation. p. 408-411. In: J. Janick (ed.),
Progress in new crops. ASHS Press, Arlington, VA.
Potential for Kenaf Improvement via Somaclonal Variation
Nancy A. Reichert and Brian S. Baldwin
- Adventitious Regeneration
- R0 and R1 Analyses for 'E41', 'G45' and 'G48'
- R1 and R2 Analyses for 'E41'
- Table 1
- Table 2
Kenaf (Hibiscus cannabinus L., Malvaceae) is a potential fiber crop
adapted to the southern United States. As part of the kenaf improvement
program at Mississippi State University, tissue culture is being employed as a
means for introduction of new or altered traits into kenaf. Regenerants often
display altered phenotypes, termed somaclonal variation (Larkin and Scowcroft,
1981). The exploitation of heritable somaclonal variants has been used in
various plant improvement strategies (Larkin and Scowcroft 1981; Evans 1989;
Larkin et al. 1989; Phillips et al. 1994) but success has been sporadic.
Our objectives were to: (1) develop an adventitious regeneration protocol for
kenaf and, (2) analyze the regenerants (R0) and subsequent progeny for trait
differences. Various traits were assessed over the course of two to three
generations (R1 and R2) and compared to control plants.
Internodal stem sections were obtained from field-grown plants of 'Everglades
41' ('E41'), surface-disinfested, then placed on a medium containing Murashige
and Skoog (1962) salts, supplemented with (per liter): 1.0 mg thiamine-HCl, 0.5
mg pyridoxine-HCl, 0.5 mg nicotinic acid, and 100 mg myo-inositol, 3% sucrose,
0.8% phytagar, plant growth regulators [0.1 mg 1-naphthaleneacetic acid (NAA)
plus 3.0 mg thidiazuron (TDZ)] with pH 5.8 for shoot initiation (Reichert and
Liu 1994). Shoots were rooted on MS medium containing no plant growth
regulators and transplanted into peat cubes for acclimatization. 'E41' R0
regenerants were transferred to the greenhouse for one week, then transplanted
into the field at the Plant Science Research Center (PSRC), at Mississippi
State Univ. in 1993. All plants were allowed to self-pollinate. R1 seeds were
harvested by plant, then germinated in peat plugs in the greenhouse and
transplanted into the field at the PSRC in 1994. R1 seedlings were compared to
'E41' seedlings produced in the same year.
In 1994, the regeneration protocol was found to be more efficient using leaf
sections. Seeds of various kenaf cultivars were germinated aseptically and
maintained in vitro via nodal sections on hormone free medium. These are
referred to as seed clonal lines (SCL). Leaf sections were explanted on
MS-based shoot induction medium containing (per liter) 0.1 mg NAA plus 3.5 mg
TDZ (Reichert and Liu 1994). We obtained 150 R0 regenerants from 'E41',
'Guatemala 45' ('G45') and 'G48' for field testing at the PSRC in June-July,
1994. In addition, 20 clonal control plants (nodal section-propagated) from
each SCL were transplanted into the field concurrently. These clonal plants
were used to determine: (1) the normal range of variation within a clonal line
as a result of environmental influences and (2) potential heterozygosity and
subsequent segregation in the original seed. (No segregation was observed.)
Both checks were necessary to determine altered phenotypes were actually due to
the leaf regeneration protocol. Additional regenerants produced after July 1st
were maintained in the greenhouse for the production of R1 seed.
In 1995, R2 seeds harvested from individual 'E41' R1 plants (generated in 1994;
from R0s planted in 1993) as well as R1 seeds harvested from each R0 regenerant
(field planted in 1994) were germinated in peat plugs in the greenhouse. Eight
days later, each line was evaluated for altered seedling phenotypes then, 15
seedlings from each line were space planted in sib rows at the PSRC. Selfed
seedlings obtained from SCL lines were space planted adjacent as controls.
Plant height, stem girth, stem color, leaf shape, days to first flower bud,
days to first open flower, and flower shape/morphology were determined in the
field in 1994 and 1995.
The leaf regeneration protocol initially developed for 'E41' was successful on
six other cultivars: E71, G4, G45, G48, G51, and Tainung 1. Adventitious
shoots were not obtained from 'Cuba 2032', 'Cubano', or 'GR2563'. However,
sufficient numbers of plantlets for field trials were generated only from E41,
G45, and G48.
R0 kenaf regenerated adventitiously from leaves displayed somaclonal variation
for stem color based on a five point color scale. Stems were classified in
discrete classes based upon the relative amount of anthocyanins present.
Individual plants fell into one of the following color classes: green, light
pink, dark pink, red, or burgundy. 'G45' regenerants displayed greater
variation in stem color than the SCL control plants. The SCL controls
displayed stem colors varying from light pink to dark pink due to environmental
effects. 'G45' R0 plants displayed stem colors spanning from green all the way
to red; displaying less or more anthocyanins in the stem than the SCL controls.
'E41' R0 plants displayed greater amounts of anthocyanins in the stem (dark
pink to red) than the corresponding SCL controls (light pink to dark pink). No
differences were detected in 'G48' between the R0 and SCL plants for stem
R0 flowers were observed for differences in flower shape/morphology. Normal
kenaf flowers have a crimson eye with five very wide light yellow flower petals
which overlap slightly. Most R0 plants displayed normal flowers, but there
were some notable exceptions. In 'E41', flowers containing narrow,
non-overlapping petals were observed. Most were composed of five petals, but
one plant consistently displayed flowers which contained six and seven petals
and one 'G48' regenerant displayed flowers with 10 petals. One 'E41' R0
regenerant had flowers that were consistently male sterile (vestigial anthers)
and one 'G48' R0 regenerant produced flowers with indehiscent anthers. One R1
plant from a 'G45' R0 line was observed to produce flowers that were pink due
to "bleeding" of the crimson eye into the petals.
R1 seedlings of all R0 plants of 'E41' displayed variation in seedling
characteristics and when mature expressed variation in days to flower as
compared with 'E41' controls. Germination of the R1 seed revealed abnormal
seedling traits. These characteristics were compared to 'E41' seedlings
generated from control plants grown in the field, adjacent to the R0 during the
same season (Table 1). There was a higher percentage of abnormal seedlings
from the R1 plants than from the 'E41' control seedlings. An albino
(chlorophyll deficient) mutation was displayed in 5% of the R1 seedlings, but
was not observed in the controls and was never detected in the millions of
seeds planted in the kenaf cultivar trial conducted in 1994 at the PSRC.
At maturity 'E41' (R1) plants were not compared to controls for stem color or
leaf shape since the controls were not SCL. Therefore, environmental
influences as well as potential outcrossing which could contribute to variation
in stem color could not be ruled out. Flowering dates for R1 plants were
compared to normal field grown 'E41' plants (Table 2). The first flower bud
appeared four weeks earlier and the first open flower appeared more than three
weeks earlier in the R1 population versus field-derived 'E41' plants. The
dates when 50% of the plants had flower buds or open flowers occurred
approximately two weeks earlier in the R1 (Table 2). No differences were
detected between R1 and field-derived 'E41' plants for plant height, stem
girth, and flower shape. R2 seed harvested from one 'E41' R1 plant displayed
the albino (chlorophyll deficient) trait upon germination at a frequency of 3%.
R0, R1, and initial R2 data confirm that the adventitious regeneration protocol
developed for kenaf results in regenerants displaying new or altered
phenotypes. Some visible traits that appeared in kenaf regenerants and
subsequent progeny (albino, crinkled cotyledons, different colored reproductive
structures, male sterility) were similar to those previously observed as single
gene mutations in progeny of maize (Zea mays L.) somaclones (Phillips et
al. 1994). Early flowering date is probably the most important variant
phenotype to come from this work. However, altered flower color, polypetally,
and male sterility are also desirable characteristics for the kenaf breeding
program. All of these altered phenotypes are indicators that heritable changes
can be made in the genome of kenaf via this leaf regeneration protocol. This
protocol has proven to be successful on seven cultivars to date. Continued
screenings will determine if any traits will be useful to the kenaf breeding
- Evans, D.A. 1989. Somaclonal variation: Genetic basis and breeding
applications. Trends Genet. 5:46-50.
- Larkin, P.J. and W.R. Scowcroft. 1981. Somaclonal variation: A novel source of
variability from cell cultures for plant improvement. Theor. Appl. Genet.
- Larkin, P.J., P.M. Banks, R. Bhati, R.I.S. Brettell, P.A. Davies. S.A. Ryan,
W.R. Scowcroft, L.H. Spindler, and G.J. Tanner. 1989. From somatic variation to
variant plants: mechanisms and applications. Genome 31:705-711.
- Phillips, R.L., S.M. Kaeppler, and P. Olhoft. 1994. Genetic instability of
plant tissue cultures: breakdown of normal controls. Proc. Nat. Acad. Sci.
- Reichert, N.A. and D. Liu. 1994. Protoplast culture and in vitro
regeneration of kenaf. Conference Proc., Sixth Int. Kenaf Assoc. p. 61-65.
Table 1. Seedling traits in control and R1 kenaf ('E41').
zControl seeds were from a commerial seedlot grown under the same
|Treatment ||n ||Normal seedling ||Empty seed ||Crinkled cotyledon ||Chlorophyll deficient ||Single cotyledon|
|Controlz ||250 ||93 ||5 ||2 ||0 ||0|
|R1y ||176 ||57 ||27 ||10 ||5 ||1|
ySeeds harvested from R0 plants in 1993 and pooled.
Table 2. Days to flower for control vs R1 kenaf ('E41').
zJune 3 was designated as day 1.
||Days after plantingz|
|Treatment ||n ||First flower bud ||50% plants w/ flower buds ||First open flower ||50% plants open flower|
|Controly ||135 ||122 ||130±6x ||136 ||149±5|
|R1w ||139 ||94 ||106±5 ||112 ||125±7|
yControl plants were initiated from seed in the field on May 19.
xNumbers ±1 SD, where applicable.
wPlants were initiated from seed on May 23 and transplanted to the field on June 3.
Last update June 17, 1997